University of Southern California Dissertations and Theses

UVRAG mediates Atg9 transport upon autophagy induction

(USC Thesis Other)

UVRAG mediates Atg9 transport upon autophagy induction

PDF

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content 1
U V R A G M E D I A T E S A T G 9 T R A N S P O R T U P O N
A U T O P H A G Y I N D U C T I O N
B y
T i a n Z ha ng
A T he s i s P r e s e nt e d t o t he
F A C U L T Y O F T H E U S C G R A D U A T E S C H O O L
U N I V E R S I T Y O F S O U T H E R N C A L I F O R N I A
I n P a r t i a l F ul f i l l m e nt of t he
R e qui r e m e nt s f or t he D e gr e e
M A S T E R O F S C I E N C E
( M O L E C U L A R M I C R O B I O L O G Y A N D I M M U N O L O G Y )
A ugus t 2014
2
A c k n ow l e d gm e n t s
F i r s t a nd f or e m os t , I w oul d l i ke t o e xpr e s s m y hone s t gr a t i t ude t o m y m e nt or , D r .
C he ngy u L i a ng f or he r e xc e l l e nt gui da nc e a nd pr ovi di ng m e t he oppor t uni t y t o do
r e s e a r c h i n our l a b. D r . L i a ng ’ s e nt hus i a s m on s c i e nc e de e pl y i ns pi r e d m e t o be a
de di c a t e d r e s e a r c he r t o pur s ue t r ut h. S he a l w a y s gi ve s m e t he be s t s uppor t a nd
e nc our a ge m e nt t ha t m ot i va t e m e t o ove r c om e di f f i c ul t i e s on r e s e a r c h a nd m a ke m e
s t r ong. I a l s o w a nt t o t ha nk m y c om m i t t e e m e m be r s D r . P i nghui F e ng a nd D r . W e i m i ng
Y ua n f or t he i r i ns t r uc t i on a nd he l p on m y t he s i s a nd de f e ns e .
M e a nw hi l e , I w oul d l i ke t o t ha nk m y s upe r vi s or D r . S ha ns ha n H e f or he r t e a c hi ng, c a r i ng
a nd pa t i e nc e t o ke e p m e pr ogr e s s i ng. S he a l w a y s c a r e f ul l y gui de s m e t o do e xpe r i m e nt s
a nd gi ve s m e pr e c i ous a dvi c e s .
A ddi t i ona l l y , I w a nt t o t ha nk m y l ove l y l a b m e m be r s : X i a ow e i Z ha ng, S a r a P i r ooz ,
Y ongf e i Y a ng, D oug O ' C onne l l , P a y a m K ha l i l z a de h, B i ny u n M a , I r e ne G hoz a l l i a nd
Y ong H e ui . W e l i ke a f a m i l y f ul l of l ove . W i t hout t he i r he l p, I w oul d ne ve r be e n a bl e t o
c om pl e t e m y r e s e a r c h w e l l a nd e nj oy t he s c i e nc e .
I a l s o w oul d l i ke t o e xpr e s s m y a ppr e c i a t i on t o D r . A xe l H . S c hont ha l , our pr ogr a m
a dvi s or , f or hi s gr e a t a dvi s e on m y s t udy a nd S i l vi na C a m pos f or he r ki ndl y he l p a l l t he
t i m e .
L a s t not t he l e a s t , I w a nt t o t ha nk m y pa r e nt s f or t he i r l ove a nd s uppor t . T he y a l w a y s gi ve
m e t he pos i t i ve e ne r gy t o f a c e c ha l l e nge s a nd s ol ve pr obl e m s .
3
T ab l e of C on t e n t s
A c know l e dgm e nt s ................................................................................................................2
A bs t r a c t ................................................................................................................................ 4
1 I n t r oduc t i on ....................................................................................................................... 5
1.1 A ut opha gy .............................................................................................................. 5
1.2 R ol e of a ut opha gy i n di s e a s e s ................................................................................ 6
1.3 T he pr oc e s s of a ut opha gy ...................................................................................... 7
1.4 A t g9 pr ot e i n a nd A t g9 t r a f f i c ki ng ........................................................................10
1.5 U V R A G ............................................................................................................... 11
2 M e t hods a nd M a t e r i a l s .................................................................................................... 13
2.1 C e l l c ul t ur e a nd t r a ns f e c t i on ................................................................................ 13
2.2 A nt i bodi e s , f l uor e s c e nt dy e s a nd ot he r r e a ge nt s . ................................................. 13
2.3 I m m unof l uor e s c e nc e a nd c onf oc a l l a s e r s c a nni ng m i c r os c opy . .......................... 13
2.4 I m m unobl ot t i ng. ...................................................................................................15
2.5 G e ne knoc kdow n by s hR N A . .............................................................................. 15
3 R e s ul t s ............................................................................................................................. 16
3.1 U V R A G i s r e qui r e d i n A t g9 t r a f f i c ki ng upon a ut opha gy i nduc t i on .................... 16
3.2 U V R A G i nt e r a c t i ons w i t h B e c l i n1 a nd P I 3P a r e r e qui r e d f or A t g9 t r a f f i c ki ng. . 18
3.3 U V R A G - m e di a t e d A t g9 t r a f f i c ki ng a f f e c t s A ut opha gos om e bi oge ne s i s . ........... 22
3.4 U V R A G - m e di a t e d A t g9 t r a f f i c ki ng i nc r e a s e s a ut opha gi c a c t i vi t y l e ve l upon
a ut opha gy i nduc t i on ................................................................................................... 24
4 D i s c us s i on ....................................................................................................................... 26
5 R e f e r e nc e s ....................................................................................................................... 29
4
A b s t r ac t
A ut opha gy i s a c e l l ul a r pr oc e s s of s e l f - di ge s t i on t o m a i nt a i n hom e os t a s i s a nd a voi d c e l l
de a t h. T he nor m a l f unc t i oni ng of a ut opha gy i s s t r ongl y l i nke d t o i t s m ol e c ul a r pa t hw a y ,
e s pe c i a l l y t he f or m a t i on of a ut opha gos om e . U ndoubt e dl y , t he f or m a t i on of
a ut opha gos om e r e l i e s on t he c ont i nuous s uppl y of m e m br a ne s our c e . A t g9, a s t he onl y
t r a ns m e m br a ne a ut opha gy pr ot e i n, pl a y s a n i m por t a nt r ol e i n m e m br a ne de l i ve r y f or
a ut opha gos om e f or m a t i on. H e r e , I i de nt i f y U V R A G , a be c l i n1- bi ndi ng a ut opha gi c
pr ot e i n, m e di a t e s A t g9 t r a f f i c ki ng upon a ut opha gy i nduc t i on t o a s s i s t t he a ut opha gos om e
bi oge ne s i s a s w e l l a s i nc r e a s e c e l l ul a r a ut opha gi c a c t i vi t y . M e a nw hi l e , t he i nt e r a c t i on
be t w e e n U V R A G a nd B e c l i n1 a l s o i s r e qui r e d f or t he A t g9 t r a f f i c ki ng upon a ut opha gy
i nduc t i on. T he s e f i ndi ngs s ugge s t U V R A G a c t s a s a n i m por t a nt r e gul a t or of A t g9
t r a ns por t t o f ur t he r pr om ot e a ut opha gos om e f or m a t i on w he n a ut opha gy i s i nduc e d.
5
1 I n t r od u c t i on
1 . 1 A u t o p h a g y
A ut opha gy , l i t e r a t e l y m e a ns s e l f - e a t i ng, i s a w or d de r i ve d f r om G r e e k r oot “ a ut o ” ( s e l f )
a nd “ pha gy ” ( e a t i ng) . A N obe l w i nne r - - C hr i s t i a n de D uve , f i r s t us e d t hi s w or d i n 1963
w he n he w or ke d on l y s os om e ( B r i nda R a vi kum a r e t a l ., 2010) .
G e ne r a l l y , a ut opha gy m a i nl y de s c r i be s a c e l l ul a r c a t a bol i c w a y of s e l f - di ge s t i on t o
m a i nt a i n t he hom e os t a s i s a nd t o a voi d c e l l de a t h. I t i s a hi ghl y r e gul a t e d pr oc e s s , r e l y i ng
on t he c ol l a bor a t i on be t w e e n a ut opha gos om e a nd l y s os om e , t o de l i ve r m odi f i e d,
unne c e s s a r y , or da m a ge d c e l l ul a r m a c r om ol e c ul e s a nd w hol e or ga ne l l e s f or hy d r ol y t i c
de gr a da t i on a nd r e c y c l i ng ( M i z us hi m a , 2007; Y or i m i t s u & K l i ons ky , 2005) .
I n m a m m a l i a n s y s t e m s , a ut opha gy unde r ba s a l l e ve l pl a y s a c ons t i t ut i ve a nd a c t i ve r ol e
i n m a i nt a i ni ng c e l l ul a r hom e os t a s i s by t ur nove r of m a c r om ol e c ul e s a nd
or ga ne l l e s ( M i z us hi m a , 2007) . A ut opha gy c a n a l s o be i nduc e d unde r nut r i e nt de pr i va t i on
a nd c e l l ul a r s t r e s s e s t o pr ovi de a s our c e of bui l di ng bl oc ks ( a m i no a c i ds , f a t t y a c i ds ,
s uga r s ) t ha t a l l ow a da pt i on of c e l l s t o a dve r s e c ondi t i ons ( B r i nda R a vi kum a r e t a l ., 2010) .
A ddi t i ona l l y , a ut opha gy pl a y s a di r e c t or i ndi r e c t r ol e i n he a l t h a nd di s e a s e , i nc l udi ng
c ont r ol of e m br y o ni c de ve l opm e nt , t i s s ue hom e os t a s i s , t um or i ge ne s i s ( L o z y & K a r a nt z a ,
2012) , c l e a r a nc e of i nt r a c e l l ul a r pa t hoge ns ( S a i t oh & A ki r a , 2010) , he a r t
di s e a s e ( N e m c he nko, C hi ong, T ur e r , L a va nde r o, & H i l l , 2011) a nd ne ur ode ge ne r a t i ve
di s e a s e s ( C ue r vo & W ong, 2010) .
6
1 . 2 R o l e o f a u t o p h a g y i n d i s e a s e s
T he r ol e of a ut opha gy i n c a nc e r i s pa r a doxi c a l . A ut opha gy c a n be i nduc e d t o pr om ot e t he
c a nc e r c e l l s ur vi va l a nd s uppor t t he m e t a bol i c r e pr ogr a m m i ng of t um or c e l l s . I t c a n a l s o
l e a d c a nc e r c e l l s t o de a t h ( L o z y & K a r a nt z a , 2012) . I n ot he r w or ds , a ut opha gy c a n be
bot h t um or i ge ni c a nd t um or s uppr e s s i ve .
A ut opha gy a c t s a s a de f e nde r a ga i ns t i nt r a c e l l ul a r pa t hoge ns . I t pl a y s a r ol e i n t he
r e c ogni t i on of a pa t hoge n a s w e l l a s t he de ve l opm e nt of a s pe c i f i c a da pt i ve i m m une
r e s pons e . A s f or i m m une c e l l s , a ut opha gy m a i nt a i ns t he hom e os t a s i s a nd m odul a t e s t he
a c t i va t i on of t he m , i nc l udi ng m a c r opha ge s , de ndr i t i c c e l l s , a nd l y m phoc y t e s ( V a l dor &
M a c i a n, 2012) . M or e ove r , s om e a ut opha gy r e l a t e d pr ot e i ns a r e i nvol ve d i n t he i nna t e
i m m une r e s pons e s ( S a i t oh & A ki r a , 2010) .
A ut opha gy f unc t i ons a s a n a da pt i ve m e c ha ni s m t o ke e p t he he a r t f r om he m ody n a m i c
s t r e s s da m a ge . R e c e nt l y , i t w a s r e por t e d t ha t c a r di a c - s pe c i f i c a ut opha gy pl a y s a r ol e i n
t he m a i nt e na nc e of c a r di a c ge om e t r y a nd c ont r a c t i l e f unc t i on ( N e m c he nko e t a l ., 2011) .
A ut opha gi c c l e a ni ng of us e l e s s c y t os ol i c c om pone nt s i s ne c e s s a r y f or r e gul a r f unc t i oni ng
of he a r t . A de l i c a t e ba l a nc e of a ut opha gy m a i nt a i ns c a r di a c hom e os t a s i s , w he r e a s a n
i m ba l a nc e l e a ds t o t he pr ogr e s s i on of he a r t f a i l ur e .
A ut opha gy ha s be e n i m pl i c a t e d i n ne ur ode ge ne r a t i ve di s e a s e s , s uc h a s P a r ki ns on ’ s
di s e a s e ( S á nc he z - P é r e z , C l a r a m ont e - C l a us e l l , S á nc he z - A ndr é s , & H e r r e r o, 2012) ,
A l z he i m e r ’ s di s e a s e ( L i , Z ha ng, & L e , 2010) a nd H unt i ngt on ’ s di s e a s e ( M a r t i ne z - V i c e nt e
e t a l ., 2010) , w hi c h a r e t he m a j or ne ur ode ge ne r a t i ve c ondi t i ons c a us i ng de m e nt i a a nd
m ove m e nt di s or de r s i n t he a gi ng popul a t i on. O ne of t he pr om i ne nt f e a t ur e s of
H unt i ngt on ’ s di s e a s e i s t he f a i l ur e of e f f i c i e nt l y t r a ppi ng t he c y t os ol i c c a r go by
7
a ut opha gos om e f or de gr a da t i on a nd t he r e f or e t he i ne f f i c i e nt t ur nove r of c e l l ul a r
c om pone nt s ( L i e t a l ., 2010) . G e ne r a l l y , t he l os s of a ut opha gy - r e l a t e d ge ne s r e s ul t s i n
a bnor m a l f unc t i on of a ut opha gy a nd a c c um ul a t i on of m i s f ol de d pr ot e i ns .
I n a w or d, a ut opha gy ha s s t r i ki ng c onne c t i ons w i t h hum a n di s e a s e a nd phy s i ol ogy . I t
s e r ve s a s a doubl e - e dge d s w or d. A l t hough a ut opha gy ba s a l l y pr ot e c t s c e l l s f r om
hom e os t a t i c i m ba l a nc e , i t i s a l s o a bl e t o he l p t he e va s i on a nd pr ogr e s s i on of c a nc e r c e l l s
a nd i nt r a c e l l ul a r pa t hoge ns t o s om e e xt e nd. U nde r s t a ndi ng t he r e gul a t or y m e c ha ni s m of
a ut opha gy m i ght ul t i m a t e l y a l l ow s c i e nt i s t s a nd c l i ni c i a ns t o de c i phe r t he pa t hoge ne s i s i n
hum a n di s e a s e a s w e l l a s f i nd t a r ge t s on pur pos e of c ur i ng s pe c i f i c di s e a s e s .
1 . 3 T h e p r o c e s s o f a u t o p h a g y
A ut opha gy c ont i nuous l y oc c ur s i n ba s a l c ondi t i ons t o m a i nt a i n hom e os t a s i s s uc h a s
pr ot e i n a nd or ga ne l l e t ur nove r a nd i s upr e gul a t e d unde r nut r i e nt s t a r va t i on or c e l l ul a r
s t r e s s c ondi t i on ( B r i nda R a vi kum a r e t a l ., 2010) . A ut opha gy i s a l s o i nc r e a s i ngl y i nduc e d
w he n c e l l a r e pr e pa r i ng f or s t r uc t ur a l r e m ode l i ng dur i ng de ve l opm e nt t r a ns i t i on, or t o r i d
t he m s e l ve s of da m a gi ng c y t opl a s m i c c om pone nt s , s uc h a s i nf e c t i on, pr ot e i n a ggr e ga t e
a c c um ul a t i on or oxi da t i ve s t r e s s ( L e vi ne & K r oe m e r , 2008) .
T he r e a r e s e ve r a l di s c r e t e s t a ge s i nvol ve d i n t he pr oc e s s of a ut opha gy . T he y a r e i ni t i a t i on
of a ut opha gy , a ut opha gos om e nuc l e a t i on a nd e l onga t i on, a ut opha gos om e m a t ur a t i on,
doc ki ng a nd f us i on of a ut opha gos om e w i t h l y s os om e a nd a ut ol y s os om e body br e a kdow n
a nd de gr a da t i on ( F i gur e 1) .
8
F i g u r e 1 T h e m o l e c u l a r p r o c e s s o f a u t o p h a g y ( M e l é n d e z & L e v i n e , 2 0 0 9 ) .
I n i t i at i on of A u t oph agy
A ut opha gos om e f or m a t i on i s hi ghl y i nduc i bl e . A ut opha gos om e f or m a t i on i s i ni t i a t e d a t
a t pha gophor e a s s e m bl y s i t e s ( P A S ) ( S uz uki e t a l ., 2001) . N ut r i e nt de pr i va t i on, a pot e nt
phy s i ol ogi c a l r e gul a t or of a ut opha gy ( K um a e t a l ., 2004) , i nduc e s a ut opha gos om e
f or m a t i on t hr ough t he i na c t i va t i on of t he ne ga t i ve r e gul a t or - - m a m m a l i a n t a r ge t of
r a pa m y c i n c om pl e x1- m T O R 1) . A nd t he n m T O R 1 di s s oc i a t e s f r om U L K c om pl e x w hi c h
r e s ul t s i n t he a c t i va t i on of U L K c om pl e x a nd i nc r e a s e d U L K 1 a nd U L K 2 ki na s e s
a c t i vi t y ( W i r t h, J oa c hi m , & T ooz e , 2013) . U s ua l l y , a bunda nc e of nut r i e nt s , or gr ow t h
f a c t or s , a c t i va t e s m T O R 1 a nd s uppr e s s e s a ut opha gy .
T he a ut opha gy - s pe c i f i c c l a s s I I I P hos pha t i dy l i nos i t ol 3- ki na s e c om pl e x ( P I 3 K C ) , a l s o
know n a s t he B e c l i n1- i nt e r a c t i ng c om pl e x, i s e qua l l y ne c e s s a r y f or i ni t i a t i ng
a ut opha gos om e f or m a t i on i n bot h y e a s t a nd m a m m a l i a n s y s t e m s . T he c or e c om pone nt s
of P I 3K C c om pl e x c ons i s t of B e c l i n1, B c l - 2 f a m i l y pr ot e i ns ( w hi c h i nhi bi t a ut opha gy ) ,
t he c l a s s I I I phos pha t i dy l i nos i t ol 3- ki na s e ( V P S 34) ( L a m b, Y os hi m or i , & T ooz e , 2013) .
9
N ot a bl y , a s B e c l i n1 i s know n t o i nt e r a c t w i t h di f f e r e nt pr ot e i ns , m ul t i pl e di s t i nc t be c l i n1-
bi ndi ng P I 3K C c om pl e xe s a r e f or m e d. O ne of t he P I 3K C c om pl e xe s i s U V r a di a t i on
r e s i s t a nc e a s s oc i a t e d ge ne ) - a s s oc i a t e d P I 3K C c om pl e x ( C . H e & L e vi ne , 2010; I t a kur a ,
K i s hi , I noue , & M i z us hi m a , 2008) . T hr ough t he a c t i va t e d U L K 1 ki na s e , V P S 34 c a n be
phos phor y l a t e d, a nd t he r e f or e a c t i vi t y of P I 3K c om pl e x c a n be e nha nc e d ( R us s e l l e t a l .,
2013) .
N u c l e at i on an d e xpan s i on of A u t oph agos om e
W he n a c t i va t i ng a nd t a r ge t i ng t o t he a ut opha gos om e i ni t i a t i on s i t e , t he U L K c om pl e x
a nd P I 3K c om pl e xe s pr om ot e t he nuc l e a t i on of doubl e - m e m br a ne s t r uc t ur e s , know n a s
i s ol a t i on m e m b r a ne or pha gop hor e by ge n e r a t i ng phos pha t i dyl i nos i t ol - 3- pho s pha t e ( P I 3 P )
( I t a kur a e t a l ., 2008) . A l s o a ddi t i ona l A T G pr ot e i ns a nd a ut opha gy - s pe c i f i c P I 3P
e f f e c t or s , s uc h a s doubl e F Y V E - c ont a i ni ng pr ot e i n1 ( D F C P 1) a nd W D - r e pe a t dom a i n
phos phoi nos i t i de - i nt e r a c t i ng ( W I P I ) pr ot e i ns a r e r e c r ui t e d f or nuc l e a t i on.
T he f ol l ow i ng e l onga t i on of i s ol a t i on m e m br a ne s t ha t de ve l op i nt o a ut opha gos om e s i s
r e gul a t e d by t w o ki nds of ubi qui t i na t i on- l i ke r e a c t i ons . T he one i s A t g16L - A t g5- A t g12-
m e di a t e d ubi qui t i na t i on- l i ke r e a c t i on. A not he r r e a c t i on i nvol ve s t he c onj uga t i on of
m i c r ot ubul e - a s s oc i a t e d pr ot e i n l i ght c ha i n 3 ( L C 3) - A t g8 t o t he l i pi d
phos pha t i dy l e t ha nol a m i ne ( P E ) ( G e ng & K l i ons ky , 2008) . T he c onj uga t i on of L C 3 t o P E
r e s ul t s i n t he c onve r s i on of c y t os ol i c L C 3- I t o a ut opha gos om e - a s s oc i a t e d L C 3- I I , vi s i bl e
a s di s pe r s e d punc t a unde r i m m unof l uor e s c e nc e a na l y s i s i ndi c a t i ng a ut opha gos om e
f or m a t i on ( B r i nda R a vi kum a r e t a l ., 2010) .
A u t oph agos om e m at u r at i on
10
T he e xpa ns i on of pha gophor e l e a ds t o t he c om pl e t e s e que s t r a t i on of c y t os ol i c
c om pone nt s , t o f or m a doubl e - m e m br a ne or ga ne l l e t e r m e d a ut opha gos om e . T hi s s t e p i s a
s i m pl e s e que s t r a t i on, a nd no de gr a da t i on oc c ur s . B y f a r , onl y L C 3- I I ha s be e n i de nt i f i e d
on t he m a t ur e a ut opha gos om a l m e m br a ne ( M i z us hi m a , 2007) . L C 3- I I ha s be e n i m pl i c a t e d
t o s e r ve a s a r e c e pt or f or a a ut opha gi c s ubs t r a t e - p62, w hi c h i s pr e f e r e nt i a l l y de gr a de d by
a ut opha gy ( N a ka i e t a l ., 2007) . p62 i s a good i ndi c a t or f or a ut opha gi c f l ux be c a us e i t i s
s e l e c t i ve l y r e c ogni z e d a nd de gr a de d by a ut opha gy .
F u s i on w i t h l y s os om e an d A u t ol y s os om e de gr adat i on
I n t he ne xt s t e p, a ut opha gos om e s f us e w i t h l y s os om e s i n m a m m a l i a n c e l l s . T he i nne r
m e m br a ne of t he a ut opha gos om e a nd t he c y t os ol i c c om pone nt s i ns i de a ut opha gos om e
a r e t he n de gr a de d by hy d r ol y t i c de gr a da t i on. T he s e de gr a di ng s t r uc t ur e s a r e of t e n c a l l e d
“ a ut ol y s os om e ” . O nc e m a c r om ol e c ul e s ha ve be e n de gr a de d i n t he l y s os om e , t he
de gr a de d m a t e r i a l l i ke a m i no a c i ds a nd f a t t y a c i ds a r e e xpor t e d t o t he c y t os ol f or
r e c y c l i ng.
1 . 4 A t g 9 p r o t e i n a n d A t g 9 t r a f f i c k i n g
E s s e nt i a l a ut opha gy pr ot e i ns a r e t he m ol e c ul a r ba s i s of a ut opha gy m a c hi ne r y . I n y e a s t ,
31 a ut opha gy r e l a t e d ge ne s w e r e de f i ne d a s be i ng di r e c t l y i nvol ve d i n t he e xe c ut i on of
a ut opha gy ( X i e & K l i ons ky , 2007) . A t l e a s t 16 m e m be r s of a ut opha gy r e l a t e d ge ne s ha ve
be e n i de nt i f i e d i n hum a n.
A m ong a ut opha gy pr ot e i ns , A t g9 i s t he onl y m ul t i s pa nni ng pr ot e i n c ont a i ni ng 6
t r a ns m e m br a ne dom a i ns a nd pl a y s c r uc i a l r ol e i n a ut opha gos om e f or m a t i on ( W e bbe r ,
Y oung, & T ooz e , 2007) . I t i s hi ghl y c ons e r ve d f r om y e a s t t o m a m m a l i a n c e l l s . I n
m a m m a l i a n c e l l s , A t g9 c y c l e s be t w e e n t he t r a ns - G ol gi ne t w or k, l a t e e ndos om e s a nd
11
a ut opha gos om e s i n r e s pons e t o t he c ha nge of c e l l ul a r s t r e s s ( W e bbe r & T ooz e , 2010) .
U nde r nor m a l c ondi t i on, f l uor e s c e nc e - t a gge d A t g9 pr e s e nt e d a l oc a t i on i n a j uxt a - nuc l e a r
r e gi on. B ut upon nut r i e nt de pr i va t i on, A t g9 s how e d a n i nc r e a s e i n pe r i phe r a l c y t opl a s m
r e gi on w hi c h w a s pos i t i ve f or L C 3 ( T a ka ha s hi e t a l ., 2011; Y oung e t a l ., 2006) .
C e l l s ut i l i z e a ut opha gy t o m a i nt a i n hom e os t a s i s by de gr a da t i on of da m a ge d or obs ol e t e
or ga ne l l e s a nd pr ot e i ns . C e nt r a l t o t hi s pr oc e s s i s t he f or m a t i on of a ut opha gos om e s , t he
doubl e m e m br a ne ve s i c l e s r e s pons i bl e f or de l i ve r i ng c y t opl a s m i c m a t e r i a l t o l y s os om e .
A nd t he s uppl y of m e m br a ne s our c e f or a ut opha gos om e i s t he pr e r e qui s i t e of a ut opha gy
pr ogr e s s i on. B ut w ha t i s t he or i gi n of a ut opha gos om a l m e m br a ne s our c e ? I t i s s t i l l a t opi c
unde r de ba t e , e ve n s e ve r a l c a ndi da t e s ha ve be e n pr opos e d a s a m e m br a ne s our c e f or
a ut opha gos om e f or m a t i on.
R e c e nt l y , t hr ough e m pl oy i ng a dva nc e d m i c r os c opy t e c hni que s c om bi ne d w i t h
bi oc he m i s t r y a ppr oa c he s , pe opl e ha ve r e c ogni z e d A t g9 ve s i c l e s ha ve be e n i m pl i c a t e d t o
be a s our c e of t he a ut opha gos om a l m e m br a ne s . O nc e t he A t g9- c ont a i ni ng ve s i c l e s a r e
pr oduc e d f r om G ol gi a ppa r a t us , a nd i m m e di a t e l y a r e a s s e m bl e d t o i s ol a t i on m e m br a ne .
A f t e r t he a s s e m bl y t o t he i s ol a t i on m e m br a ne , t he A t g9 ve s i c l e s a r e s ubs e que nt l y
i nc or por a t e d i nt o t he a ut opha gos om a l out e r m e m br a ne ( Y a m a m ot o e t a l ., 2012) .
1 . 5 U V R A G
T he ul t r a vi ol e t ( U V ) r a di a t i on r e s i s t a nc e - a s s oc i a t e d ge ne ( U V R A G ) w a s or i gi na l l y
i de nt i f i e d a s a n a ut opha gy pr ot e i n, a nd ha s be e n pr e di c t e d t o be hom ol ogous t o V P S 38 i n
y e a s t ( I t a kur a e t a l ., 2008) . W hi l e U V R A G pl a y s i m por t a nt a ut opha gi c r ol e s , i t s t i l l
pos s e s s e s ot he r va r i ous pr ope r t i e s t ha t m a ke i t m y s t e r i ous but i nt e r e s t i ng. I n ve s t i ga t i ons
ha ve s how n U V R A G a l s o s e r ve t o m oni t or e ndoc y t i c m e m br a ne t r a f f i c ki ng ( L i a ng e t a l .,
12
2008) , m a i nt a i n c hr om os om a l s t a bi l i t y ( Z ha o e t a l ., 2012) , s uppr e s s t um or
pr ogr e s s i on ( L i a ng e t a l ., 2006) , r e gul a t e a popt os i s dur i ng c he m ot he r a py ( Y i n e t a l ., 2011)
a nd m e di a t e G ol gi - E R r e t r ogr a de t r a ns por t ( S . H e , O ' C onne l l , Z ha ng, Y a ng, & L i a ng,
2014) .
T he m ul t i f unc t i on of U V R A G m a i nl y a t t r i but e s t o i t s f i ve f unc t i ona l dom a i ns : t he
pr ol i ne - r i c h ( P R ) dom a i n ( r e s i due s 1- 41) , t he l i pi d- bi ndi ng C 2 dom a i n ( r e s i due s 42- 147) ,
t he be c l i n- 1 bi ndi ng c oi l e d- c oi l dom a i n ( C C D , r e s i due s 200- 269) , t he R I N T - 1- E R - t e t he r
bi ndi ng dom a i n ( r e s i due s 270- 442) , a nd t he C - t e r m i na l dom a i n ( r e s i due s 443- 699) .
O ur r e c e nt w or k s how e d t ha t U V R A G i nt e r a c t e d w i t h P I 3P t hr ough i t s C 2 dom a i n a nd
w a s s ubs e que nt l y l oc a l i z e d t o E R . S pe c i f i c a l l y , t w o c ons e r ve d r e s i due s ( L y s 78 a nd A r g
82) i n C 2 dom a i n w e r e i de nt i f i e d a s P I 3P bi ndi ng s i t e s . A doubl e a l a ni ne s ubs t i t ut i on
( K 78A / R 82A ) s uppr e s s e d phos phol i pi d bi ndi ng of U V R A G - C 2. F ur t he r m or e , P I 3P -
bound U V R A G f unc t i one d a s a s c a f f ol d f or t he r e c r ui t of R I N T 1- E R - t e t he r , w hi c h
m e di a t e d t he C O P I - c oa t e d c a r go de l i ve r y unde r phy s i ol ogi c a l c ondi t i on. E i t he r de l e t i on
of R I N T 1 bi ndi ng D om a i n of U V R A G or t he doubl e A na l i ne s ubs t i t ut i on w oul d di s r upt
G ol gi - E R t r a ns por t a nd t he r e f or e r ui ne d G ol gi s t a bi l i t y ( S . H e e t a l ., 2013) .
I n a ddi t i on, our pr e vi ous s t udy i ndi c a t e d t ha t U V R A G w or ke d i n c onc e r t w i t h B e c l i n1-
bi ndi ng P I 3K C c om pl e x t o pr om ot e a ut opha gos om e f or m a t i on ( L i a ng e t a l ., 2006) . B ut
t he e xa c t m e c ha ni s m of U V R A G - m e di a t e d a ut opha gy a c t i va t i on ha s n' t be e n i de nt i f i e d
y e t .
13
2 M e t h od s an d M at e r i al s
2 . 1 C e l l c u l t u r e a n d t r a n s f e c t i o n
293T a nd H e L a c e l l s w e r e c ul t ur e d i n D ul be c c o ’ s m odi f i e d E a gl e ’ s m e di um ( D M E M )
w i t h 10% f e t a l bovi ne s e r um ( F B S ; I n vi t r oge n) , 2m M L - G l ut a m i ne ( G i bc o) a nd 50ug/ m l
pe ni c i l l i n- s t r e pt om y c i n ( G i bc o- B R L ) . T he c e l l s w e r e m a i nt a i ne d i n t he i nc uba t or w i t h a n
a t m os phe r e s e t t o 5% C O 2 a nd 37° C . T r a ns f e c t i on w e r e pe r f or m e d w i t h F uG E N E 6 H D
( R oc he ) or L i pof e c t a m i ne 2000 ( I n vi t r oge n) , f ol l ow i ng t he i r m a nuf a c t ur e r ’ s i ns t r uc t i ons .
2 . 2 A n t i b o d i e s , f l u o r e s c e n t d y e s a n d o t h e r r e a g e n t s .
T he f ol l ow i ng a nt i bodi e s w e r e us e d i n t hi s s t udy : pol y c l ona l r a bbi t a nt i - U V R A G ( U 7058,
S i gm a - A l dr i c h) a t 1: 1,000; m onoc l ona l m ous e a nt i - U V R A G ( S A B 4200005, c l one
U V R A G - 11, S i gm a - A l dr i c h) a t 1: 200; pol y c l ona l goa t a nt i - R I N T - 1 ( S a nt a - C r uz ) a t
1: 500; m onoc l ona l m ous e a nt i - be c l i n- 1 ( 612113, c l one 20, S i gm a ) a t 1: 500 ; m onoc l ona l
m ous e a nt i - a c t i n ( s c - 47778, c l one C 4, S a nt a - C r uz ) a t 1: 5,000; pol y c l ona l r a bbi t a nt i - A t g9
a t 1: 100 ( G e ne T e x) ; m onoc l ona l m ous e a nt i - L C 3 a t 1: 130 ( C os m o B i o, J a pa n) ;
m onoc l ona l m ous e a nt i - p62 a t 1: 3,000 ( M B L , J a pa n) ; m onoc l ona l m ous e a nt i - L C 3 a t
1: 500 ( C A C - C T B - L C 3- 2- I C ; c l one : L C 3- 1703; C os m o B i o U S A ) ; m onoc l ona l a nt i - F l a g
( F 3165, c l one M 2, S i gm a ) a t 1: 2,000; f l uor e s c e nt l y l a be l e d s e c onda r y a nt i body
c onj uga t e s , pur c ha s e d f r om M ol e c ul a r P r obe s ( I n vi t r oge n) .
2 . 3 I m m u n o f l u o r e s c e n c e a n d c o n f o c a l l a s e r s c a n n i n g m i c r o s c o p y .
I m m unof l uor e s c e nc e m i c r os c opy w a s c a r r i e d out a s f ol l ow i ng de s c r i pt i ons . C e l l s w e r e
pl a t e d on c ove r s l i ps a nd f i xe d w i t h 4% pa r a f or m a l de hy d e ( 30 m i n a t r oom t e m pe r a t ur e ) .
A f t e r f i xa t i on, c e l l s w e r e pe r m e a bi l i z e d w i t h 0.2% T r i t on X - 100 f or 5 m i n a nd bl oc ke d i n
14
phos pha t e buf f e r e d s a l i ne ( P B S ) w i t h 10% goa t s e r um ( G i bc o- B R L ) f or 1 hour ( h) .
A nt i s e r um or pur i f i e d pr i m a r y a nt i body s t a i ni ng w a s c a r r i e d out i n P B S w i t h 1% goa t
s e r um f or 1 h a t r oom t e m pe r a t ur e , or ove r ni ght a t 4 ° C . C e l l s w e r e t he n e xt e ns i ve l y
w a s he d w i t h P B S a nd i nc uba t e d w i t h di l ut e d A l e xa 488- , A l e xa 594- a nd/ or A l e xa 633-
c onj uga t e d s e c onda r y a nt i bodi e s i n P B S w i t h 1% goa t s e r um f or 1 h, f ol l ow e d by D A P I
( 4, 6 - di a m i di no- 2- phe ny l i ndol e ) s t a i ni ng f or 30 m i n. C e l l s w e r e m ount e d us i ng
V e c t a s hi e l d ( V e c t or L a bor a t or i e s ) . T o s t a i n e ndoge nous m e m br a ne - a s s oc i a t e d pr ot e i ns ,
c e l l s w e r e f i r s t pe r m e a bi l i z e d w i t h 0.05% s a poni n f or 1 m i n t o e xt r a c t c y t os ol be f or e
f i xi ng. T he c e l l s w e r e t he n f i xe d, pe r m e a bi l i z e d a nd pr oc e s s e d f or i m m unof l uor e s c e nc e
m i c r os c opy a s de s c r i be d a bove . C onf oc a l i m a ge s w e r e a c qui r e d us i ng a N i kon E c l i ps e
C 1 l a s e r - s c a nni ng m i c r os c ope ( N i kon) , f i t t e d w i t h a 60× N i kon obj e c t i ve ( P L A P O , 1.4
N A ) , a nd N i kon i m a ge s of t w a r e . I m a ge s w e r e c ol l e c t e d a t 512× 512 pi xe l r e s ol ut i on. T he
s t a i ne d c e l l s w e r e opt i c a l l y s e c t i one d i n t he z a xi s . F or m ul t i c ha nne l i m a gi ng, f l uor e s c e nt
s t a i ni ng w a s i m a ge d s e que nt i a l l y i n l i ne - i nt e r l a c e m ode s , t o e l i m i na t e c r os s t a l k be t w e e n
t he c ha nne l s . T he s t e p s i z e i n t he z a xi s va r i e d f r om 0.2 t o 0.5 m m t o obt a i n 16 s l i c e s pe r
i m a ge d f i l e . A l l e xpe r i m e nt s w e r e i nde pe nde nt l y r e pe a t e d s e ve r a l t i m e s .
T he N i kon N I S - E l e m e nt s i m a gi ng a na l y s i s s of t w a r e w a s us e d f or t he c o- l oc a l i z a t i on
a na l y s e s . I n t he a na l y s i s , c onf oc a l i m a ge s of doubl e - s t a i ne d s e c t i ons w e r e f i r s t s ubj e c t e d
t o ba c kgr ound c or r e c t i on. M a nde r ’ s ove r l a p c oe f f i c i e nt s w e r e c a l c ul a t e d a nd us e d t o
obt a i n t he c o- l oc a l i z a t i on va l ue s a s pe r c e nt a ge s of U V R A G ove r l a ppi ng w i t h t he pr ot e i n
of i nt e r e s t ( f or e xa m pl e , di f f e r e nt or ga ne l l e m a r ke r s ) , or a s pe r c e nt a ge s pe r c e l l of A t g9
t ha t c o- l oc a l i z e d w i t h t he L C 3 punc t a f or 50–100 c e l l s . V a l ue s i ndi c a t e t he m e a n ± s .d. of
a t l e a s t t hr e e i nde pe nde nt e xpe r i m e nt s .
15
F or t he i m a gi ng e xpe r i m e nt of ge ne knoc kdow n a nd/ or c om pl e m e nt a t i on, t he c e l l
s a m pl e s w e r e a l l oc a t e d a c c or di ng t o t he r e por t e r ( f or e xa m pl e , G F P ) or t he s pe c i f i c
pr ot e i n s t a i ni ng. A ppr oxi m a t e l y 50–200 c e l l s f r om 10 t o 20 r a ndom l y a nd bl i ndl y c hos e n
hi gh- pow e r f i e l ds ( 20 or 60× ) w e r e e va l ua t e d f or i m a gi ng a na l y s i s .
2 . 4 I m m u n o b l o t t i n g .
F or i m m unobl ot t i ng, de na t ur e d pr ot e i ns w e r e r un on a 6% - 15% S D S - P A G E ge l ,
ge ne r a l l y f or 30m i n a t 60V a nd a not he r 100m i n f or 110v. T he S D S - P A G E ge l w a s
t r a ns f e r r e d t o a P V D F m e m br a ne ( B i o- R a d) f or 300m A f or 90m i n. M e m br a ne s w e r e
bl oc ke d w i t h 5% non- f a t m i l k a t r oom t e m pe r a t ur e . T he n t he m e m br a ne s w e r e w a s he d
w i t h P B S T onc e a nd pr obe d w i t h t he pr i m a r y a nt i body ove r ni ght . T he m e m br a ne w a s
w a s he d w i t h P B S T f or 3 t i m e s , 10m i n pe r t i m e . T he n t he m e m br a ne s w e r e i nc uba t e d
w i t h i ndi c a t e d H or s e r a di s h pe r oxi da s e ( H R P ) - c onj uga t e d goa t s e c onda r y a nt i bodi e s
( 1: 5,000, I n vi t r oge n) f or 45 m i n. T he m e m br a ne s w e r e w a s he d w i t h P B S T f or 3 t i m e ,
e a c h t i m e 10 m i n . I m m uno bl ot t i ng w a s a c hi e ve d w i t h t he H y g l o c he m i l um i ne s c e nc e
r e a ge nt ( D e nvi l l e S c i e nt i f i c ) , a nd de t e c t e d by a F uj i E C L m a c hi ne ( L A S - 3000) .
2 . 5 G e n e k n o c k d o w n b y s h R N A .
A l l s hR N A s w e r e pur c ha s e d f r om O pe n B i os y s t e m s . T he s hR N A s e que nc e t a r ge t i ng
hum a n uvr a g i s : 5’ - A C G G A A C A T T G T T A A T A G A A - 3’ ; t a r ge t i ng hum a n R I N T - 1 i s :
5’ - C C A A T T G G A T T T C A A G T T A T - 3’ ; t a r ge t i ng hum a n be c l i n 1 i s : 5’ -
A A G A T C C T G G A C C G T G T C A C C - 3’ .
16
3 R e s u l t s
3 . 1 U V R A G i s r e q u i r e d i n A t g 9 t r a f f i c k i n g u p o n a u t o p h a g y i n d u c t i o n
T he pr e vi ous s t udy ha s de m ons t r a t e d t ha t U V R A G i s a n e s s e nt i a l c om pone nt of t he
B e c l i n1- P I 3 K C 3 l i pi d ki na s e c om pl e x , w hi c h i s a n i m por t a nt s i gna l i ng c he c kpoi nt f or
a ut opha gy a nd t um or c e l l gr ow t h ( L i a ng, F e ng, K u, O h, & J ung, 2007) . I n de t a i l , U V R A G
i nt e r a c t s B e c l i n1 t hr ough i t s c oi l e d- c oi l dom a i n ( C C D ) a nd t he e xpr e s s i on of U V R A G
i nc r e a s e t he de gr e e of a ut opha gos om e f or m a t i on ( L i a ng e t a l ., 2006) . B ut t he e xa c t
pa t hw a y i n U V R A G - m e di a t e d a ut opha gos om e f or m a t i on ha s not be e n i de nt i f i e d y e t .
C ons i de r i ng t ha t A t g9 i s a n e s s e nt i a l r e s our c e de l i ve r e d t o a ut opha gos om e m e m br a ne t o
he l p t he f or m a t i on of a ut opha gos om e , a n i nt e r e s t i ng que s t i on ha s r i s e n . W he t he r i s
U V R A G r e qui r e d i n A t g9 t r a f f i c ki ng f or a ut opha gos om e f or m a t i on upon a ut opha gy
i nduc t i on?
T o a ddr e s s t hi s que s t i on, t he di s t r i but i on of e ndoge nous A t g9 i n c ont r ol knoc kdow n a nd
i n U v r ag knoc kdow n w a s c om pa r e d bot h i n nor m a l c ondi t i on a nd i n a ut opha gy - i nduc e d
c ondi t i ons . A s s how n i n F i gur e 2, bot h i n c ont r ol knoc kdow n c e l l s a nd i n U v r ag
knoc kdow n c e l l s , e ndoge nous A t g9 pr ot e i ns i n gr e e n f l uor e s c e nc e w e r e de t e c t e d m a i nl y
i n t he j uxt a - nuc l e a r r e gi on ( G ol gi - l i ke s t r uc t ur e ) unde r nor m a l c ul t ur e c ondi t i ons .
H ow e ve r , upon R a pa m y c i n- i nduc e d a ut opha gy , t he j uxt a - nuc l e a r c onde ns e d s t r uc t ur e s of
A t g9 w e r e s i gni f i c a nt l y r e duc e d a nd m os t of t he A t g9- pos i t i ve punc t a t e s t r uc t ur e s
di s pe r s e d i n t he pe r i phe r a l c y t opl a s m . I n c ont r a s t , knoc kdow n of U v r ag a br oga t e d
R a pa m y c i n- i nduc e d t r a ns l oc a t i on of A t g9 t o pe r i phe r a l c y t opl a s m . S i m i l a r r e s ul t s w e r e
17
obs e r ve d i n H B S S s t a r va t i on- i nduc e d a ut opha gy , w hi c h i s m T O R - de pe nde nt a ut opha gy ,
a nd S M E R 28- i nduc e d a ut opha gy , w hi c h i s t he m T O R - i nde pe nde nt a ut opha gy .
F i g u r e 2 . U V R A G i n t e r r u p t e d A t g 9 t r a n s l o c a t i o n u p o n a u t o p h a g y i n d u c t i o n .
T he knoc k dow n H e L a c e l l s w e r e i nc uba t e d i n c om pl e t e m e di um ( U T ) , s t a r va t i on
m e di um ( H B S S , 2 hr ) , or t r e a t e d w i t h r a pa m y c i n ( 100nM , 2hr ) or S M E R 28 ( 50 μ M , 2hr ) .
T he di s t r i but i on pa t t e r n of e ndoge nous A t g9 w a s a na l y z e d by c onf oc a l m i c r os c opy .
18
3 . 2 U V R A G i n t e r a c t i o n s w i t h B e c l i n 1 a n d P I 3 P a r e r e q u i r e d f o r A t g 9
t r a f f i c k i n g .
U V R A G , w hi c h w a s or i gi na l l y i de nt i f i e d a s a n a ut opha gy pr ot e i n, i s a m ul t i f unc t i ona l
pr ot e i n. I t i nt e r a c t s w i t h di f f e r e nt pa r t ne r s t o c onduc t m ul t i pl e f unc t i ons t hr ough i t s
s e ve r a l f unc t i ona l dom a i ns . I n or de r t o f ur t he r s t udy how U V R A G i s r e qui r e d i n A t g9
t r a f f i c ki ng, I f i r s t i de nt i f i e d w hi c h f unc t i ona l dom a i n of U V R A G i s r e qui r e d f or A t g9
T r a f f c ki ng .
U nde r t he nor m a l c ondi t i on, s i m i l a r t o knoc kdow n c ont r ol gr oup, e ndoge nous A t g9
pr ot e i ns i n gr e e n f l uor e s c e nc e w e r e a l l ke pt i n G ol gi - l i ke s t r uc t ur e i n U V R A G - de pl e t e d
gr oups . U nde r t he r a pa m y c i n- i nduc e d a ut opha gy , f or c ont r ol knoc kdow n gr oup, A t g9
s how e d di s pe r s e d s t r uc t ur e s t hr oughout t he c y t opl a s m ( F i gur e 3a ) . R e m a r ka bl y , t he
de pl e t i on of U V R A G l e a de d t o t he f a i l ur e of A t g9 t r a ns l oc a t i on. H ow e ve r , t he
di s t r i but i on f a i l ur e of A t g9 w a s r e s c ue d by w i l d- t y p e U V R A G a nd U V R A G
∆ 2 7 0 - 4 4 2
m ut a nt , but not by U V R A G
∆ C C D
m ut a nt a nd U V R A G
K 7 8 A / R 8 2 A
m ut a nt . I n or de r t o f ur t he r
c onf i r m t he r e s ul t , a not he r t w o a ut opha gy i nduc e r s - H B S S a nd S M E R 28- w e r e
i m pl e m e nt e d i n t he s a m e a s s a y t o i nduc e a ut opha gy . S i m i l a r r e s ul t s w e r e obs e r ve d
( F i gur e 3a ) . C ons i s t e nt l y , t he qua nt i t a t i ve da t a s how s a gr e a t r e duc t i on of pe r c e nt a ge of
c e l l s w i t h A t g9 t r a ns l oc a t i on i n U V R A G
K 7 8 A / R 8 2 A
m ut a nt c om pl e m e nt a t i on gr oup a s w e l l
a s U V R A G
∆ C C D
m ut a nt c om pl e m e nt a t i on gr oup, c om pa r e d w i t h U V R A G w i l d t y p e
c om pl e m e nt a t i on gr oup a nd U V R A G
∆ 2 7 0 - 4 4 2
m ut a nt c om pl e m e nt a t i on gr oup ( F i gur e 3b) .
19
a
b c
F i g u r e 3 C C D d o m a i n o f U V R A G a n d P I 3 P b i n d i n g w i t h U V R A G a r e r e q u i r e d f o r A t g 9
t r a n s l o c a t i o n .
a) H e L a c e l l s w e r e s t a bl y t r a ns f e c t e d w i t h c ont r ol s hR N A ( f i r s t - r ow ) or U V R A G s hR N A
( s e c ond t o s i xt h r ow s ) . T he U V R A G - de pl e t e d c e l l s w e r e t he n t r a ns i e nt l y t r a ns f e c t e d w i t h
e m pt y ve c t or ( s e c ond r ow ) , F l a g- U V R A G ( t hi r d r ow ) , F l a g- U V R A G
△
2 7 0 - 4 4 2
m ut a nt
( f or t h r ow ) , F l a g- U V R A G
△
C C D
m ut a nt ( f i f t h r ow ) , or F l a g- U V R A G
K 7 8 A / R 8 2 A
m ut a nt
( s i xt h r ow ) . A f t e r t r a ns i e nt t r a ns f e c t i on, c e l l s w e r e i nc uba t e d i n c om pl e t e m e di um
( unt r e a t e d, U T ) , s t a r va t i on m e di um ( H B S S , 2hr ) , or t r e a t e d w i t h r a pa m y c i n ( 50m M ,2 hr )
or S M E R 28 ( 50 μ M , 2h) . T he di s t r i but i on pa t t e r n of e ndoge nous A t g9 ( gr e e n) i n t he
U V R A G w i l d- t y p e a nd m ut a nt t r a ns duc e d c e l l s w a s a na l y z e d by i m m unof l uor e s c e nc e .
A l s o t he f l a g- t a gge d U V R A G w i l d t y p e a nd m ut a nt s w e r e a na l y z e d by
20
i m m unof l uor e s c e nc e ( r e d) . A s t e r i s ks de not e c onde ns e d A t g9 i n U V R A G - de pl e t e d H e L a
c e l l s a nd a r r ow s de not e t he c e l l s c om pl e m e nt e d w i t h w i l d t y p e or m ut a nt U V R A G . S c a l e
ba r s , 10μ m . b ) T he di s t r i but i on of c e l l s w i t h di s pe r s e d A t g9 f oc i w a s qua nt i f i e d. F or e a c h
s a m pl e , 500 c e l l s w e r e c ount e d. c ) T he e xpr e s s i on of e ndoge nous a nd e xoge nous
U V R A G a nd a c t i n w a s de t e r m i ne d by i m m unobl ot t i ng. D a t a r e pr e s e nt m e a n ± s .d. ；
n= 500 s hR N A - t r a ns f e c t e d c e l l s obt a i ne d f r om t hr e e i nde pe nde nt e xpe r i m e nt s ; n.s ., not
s i gni f i c a nt ; **P < 0.01; ***P < 0.001.
A s w e kno w , U V R A G C C D dom a i n i s r e s p ons i bl e f o r B e c l i n 1 bi nd i ng ( L i a ng e t a l . , 200 6)
a nd U V R A G 270- 442 dom a i n m e di a t e s R I N T 1- E R t e t he r bi ndi ng ( S . H e e t a l ., 2013) . I n
or de r t o de t e c t i f i nt e r a c t i on be t w e e n U V R A G a nd B e c l i n1 or be t w e e n U V R A G a nd
R I N T 1 pl a y s a r ol e i n A t g9 t r a f f i c ki ng, t he e f f e c t of e ndoge nous B e c l i n1, R I N T 1 a nd
U V R A G w a s a na l y z e d on a ut opha gy - i nduc e d A t g9 t r a f f i c ( F i gur e 4) .
I n c onc e r t w i t h t he c om pl e m e nt a t i on a s s a y , U V R A G - a nd B E C N 1- de pl e t e d c e l l s di dn ’ t
pr e s e nt A t g 9 punc t a f or m a t i on s ur r oundi ng pe r i phe r a l c y t opl a s m a f t e r a ut opha gy
i nduc t i on. I n c ont r a s t , s i m i l a r w i t h knoc kdow n c ont r ol gr oup, R I N T 1- de pl e t e d c e l l s
e xhi bi t e d di s pe r s e d t r a ns l oc a t i on of A t g9, w hi c h m e a ns t he de pl e t i on of R I N T 1 ha s no
obs e r va bl e i nhi bi t i on f or A t g9 t r a ns l oc a t i on.
21
a
b c
F i g u r e 4 E f f e c t o f U V R A G , B e c l i n 1 a n d R I N T 1 o n a u t o p h a g y - i n d u c e d A t g 9 t r a f f i c .
a ) C o n t r o l - , U V R A G - , B e c l i n 1 - a n d R I N T 1 - K n o c k d o w n H e L a c e l l s w e r e i n c u b a t e d i n c o m p l e t e
m e d i u m ( U T ) , s t a r v a t i o n m e d i u m ( H B S S , 2 h r ) , o r t r e a t e d w i t h r a p a m y c i n ( 1 0 0 m M , 2 h r ) o r
S M E R 2 8 ( 5 0 m M , 2 h r ) . T h e d i s t r i b u t i o n o f e n d o g e n o u s A t g 9 w a s a n a l y z e d b y c o n f o c a l
m i c r o s c o p y . C e l l n u c l e u s w e r e s t a i n e d b y D A P I a s b l u e . S c a l e b a r s , 1 0 μ m . b ) T h e d i s t r i b u t i o n o f
c e l l s w i t h d i s p e r s e d A t g 9 f o c i w a s c o u n t e d . F o r e a c h s a m p l e , 5 0 0 c e l l s w e r e c o u n t e d .
c ) I m m u n o b l o t t i n g o f U V R A G , B e c l i n 1 a n d R I N T 1 a r e s h o w n . D a t a r e p r e s e n t m e a n ± S D ； n = 5 0 0
s h e R N A - t r a n s f e c t e d c e l l s o b t a i n e d f r o m t h r e e i n d e p e n d e n t e x p e r i m e n t s .
22
3 . 3 U V R A G - m e d i a t e d A t g 9 t r a f f i c k i n g a f f e c t s A u t o p h a g o s o m e
b i o g e n e s i s .
B e c a us e of t he e s s e nt i a l a s s oc i a t i on of A t g9 w i t h m e m br a ne , i t ha s be e n i m pl i c a t e d t ha t
A t g9 i s i nvol ve d i n de l i ve r i ng m e m br a ne s our c e t o a ut opha gos om e ( M a r i e t a l ., 2010) . I n
a not he r w or d, A t g9 t r a ns por t i s f unc t i ona l l y l i nke d t o a ut opha gos om e bi oge ne s i s .
C ons i de r i ng t he U V R A G m e di a t i on of A t g9 t r a f f i c ki ng, I e xa m i ne d w he t he r U V R A G -
m e di a t e d A t g9 t r a f f i c ki ng f unc t i ona l l y a f f e c t s a ut opha gos om e bi oge ne s i s .
C ons i s t e nt l y , U V R A G a nd B E C N 1 de pl e t i on pr e ve nt e d A t g9 ve s i c l e s f or m a t i on. B ot h
t he t ot a l num be r of a ut opha gi c ve s i c l e s a nd t he ove r l a ppi ng A t g9 s t a i ni ng w i t h L C 3
punc t a w e r e l a r ge l y s uppr e s s e d. I n c ont r a c t , de pl e t i on of R I N T 1 ha d no obs e r va bl e e f f e c t
on A t g9 t r a ns l oc a t i on a s w e l l a s t he ove r l a ppi ng A t g9 s t a i ni ng w i t h L C 3 punc t a ( F i gur e
5) .
23
a
b c
F i gur e 5 E f f e c t of knoc kdow n of U V R A G , B E C N 1 a nd R I N T 1 on t he t r a ns l oc a t i on of
A t g9 t o t he L C 3 l a be l e d a ut opha gos om e s dur i ng a ut opha gy .
a) C ont r ol - , U V R A G - , B E C N 1 - or R I N T 1 - knoc kdow n H e L a c e l l s w e r e i nc uba t e d i n
c om pl e t e m e di um ( nor m a l c ondi t i on) , s t a r va t i on m e di um ( H B S S , 2 h) , or t r e a t e d w i t h
r a pa m y c i n ( 100 nM , 2 h) or S M E R 28 ( 50 μ M , 2 h) . T he c e l l s w e r e t he n s t a i ne d f or
e ndoge nous A t g9 ( gr e e n) a nd L C 3 ( r e d) f ol l ow e d by c onf oc a l m i c r os c opy a na l y s i s ( t op
a nd bot t om l e f t pa ne l s ) . S c a l e ba r s , 10 μ m . b ) T he pe r c e nt a ge of A t g9 ve s i c l e s c o-
l oc a l i z e d w i t h L C 3+ - a ut opha gi c punc t a w a s qua nt i f i e d ( bot t om m i ddl e pa ne l ) . c )
E ndoge nous U V R A G , B e c l i n 1 a nd R I N T - 1 pr ot e i n e xpr e s s i on a r e s how n ( bot t om r i ght ) .
24
D a t a r e pr e s e n t m e a n ± s .d.; n = 200 c e l l s obt a i ne d f r o m t hr e e i nde pe n de nt e xp e r i m e n t s . N S ,
not s i gni f i c a nt ; *P < 0.05; **P < 0.01; ***P < 0.001.
3 . 4 U V R A G - m e d i a t e d A t g 9 t r a f f i c k i n g i n c r e a s e s a u t o p h a g i c a c t i v i t y
l e v e l u p o n a u t o p h a g y i n d u c t i o n
I n a gr e e m e nt w i t h t he f i ndi ngs t ha t A t g9 t r a f f i c ki ng i s f unc t i ona l l y l i nke d t o
a ut opha gos om e s , t he pr oduc t i on of L C 3- I I w a s gr e a t l y i nc r e a s e d i n t he U V R A G w i l d-
t y p e a nd U V R A G
∆ 2 7 0 - 4 4 2
e xpr e s s i ng c e l l s , c om pa r i ng w i t h t ha t i n t he U V R A G
∆ C C D
or
U V R A G
K 7 8 A / R 8 2 A
e xpr e s s i ng c e l l s ( F i gur e 6) .
H ow e ve r , t he i nc r e a s e of a ut opha gos om e num be r s a nd L C 3- I I e xpr e s s i on doe s not
ne c e s s a r i l y m e a n t he e nha nc e m e nt of a ut opha gi c f l ux. A ut opha gy a c t i vi t y i s r e pr e s e nt e d
by t he c om pl e t i on of t he e nt i r e pa t hw a y i nc l udi ng t he f us i on of a ut opha gos om e w i t h
l y s os om e a s w e l l a s t he c a r go de gr a da t i on w i t hi n l y s os om e . T he r e a r e a l s o ot he r
pos s i bi l i t i e s f or t he a c c um ul a t i on of a ut opha gos om e t ha t t he a ut opha gos om e f us i on w i t h
l y s os om e or de gr a da t i on w i t hi n l y s os om e m i ght be i nhi bi t e d.
T o ve r i f y t ha t t he e nha nc e d L C 3- I I e xpr e s s i on r e pr e s e nt s t he i nc r e a s e of a ut opha gi c
a c t i vi t y r a t he r t ha n t he i m pa i r e d de gr a da t i on, a not he r a ut opha gi c s ubs t r a t e p62 w a s
de t e c t e d. E xpe c t e dl y , t he l e ve l s of p62 i n U V R A G w i l d t y p e gr oup a nd i n U V R A G
∆ 2 7 0 -
4 4 2
m ut a nt gr oup m a r ke dl y r e duc e d, c om pa r e d w i t h ve c t or , U V R A G
∆ C C D
m ut a nt gr oup
a nd U V R A G
K 7 8 A / R 8 2 A
gr oup ( F i gur e 6) .
25
F i gur e 6 A ut opha gy a c t i vi t y of t he U V R A G m ut a nt s . T he 293T c e l l s e xpr e s s i ng e m pt y
ve c t or , F l a g- t a gge d w i l d- t y p e U V R A G or i t s m ut a nt s a s i ndi c a t e d, w e r e c ul t ur e d i n
c om pl e t e m e di um or t r e a t e d w i t h S M E R 28 ( 50 μ M ) . T he c e l l l y s a t e s w e r e s ubj e c t e d t o
i m m unobl ot t i ng w i t h L C 3, p62, F l a g, a nd a c t i n a nt i bodi e s . D e ns i t om e t r i c qua nt i f i c a t i on
of t he L C 3- I I / L C 3- I r a t i os unde r t he i ndi c a t e d c ondi t i ons i s s how n a t t he bot t om of t he
L C 3 bl ot . S i m i l a r r e s ul t s w e r e obt a i ne d f r om t hr e e i nde pe nde nt e xpe r i m e nt s .
26
4 D i s c u s s i on
I n t he pr e s e nt s t udy , I ha ve de m ons t r a t e d t ha t U V R A G , a m ul t i f unc t i ona l pr ot e i n, i s
i nvol ve d i n t he t r a ns por t of A t g9 upon a ut opha gy i nduc t i on, a nd f ur t he r de t e r m i ne d
U V R A G - m e di a t e d A t g9 t r a f f i c ki ng a f f e c t s a ut opha gos om e bi oge ne s i s a nd t he ove r a l l
a ut opha gi c f l ux.
A ut opha gos om e f or m a t i on i s t he c e nt r a l t o a ut opha gy pr oc e s s ( X i e & K l i ons ky , 2007) .
U V R A G pr e vi ous l y ha s be e n i de nt i f i e d t o be a n i m por t a nt pl a y e r i n t he pr om ot i on of
a ut opha gos om e f or m a t i on t hr ough t he i nt e r a c t i on w i t h B e c l i n1 ( L i a ng e t a l ., 2006) .
H ow e ve r , unde r t he pr oc e s s of a ut opha gos om e f or m a t i on, t he e xa c t m e c ha ni s m of how
U V R A G pr om ot e s a ut opha gos om e f or m a t i on ha s not be e n de t e r m i ne d y e t . C ons i de r i ng
A t g9 i s t he onl y know n t r a ns m e m br a ne pr ot e i n r e qui r e d f or a ut opha gy a nd ha s be e n
i m pl i c a t e d t o be m e m br a ne s uppl i e r t o t he pr e a ut opha gos om e s t r uc t ur e s a nd
a ut opha gos om e s ( O r s i e t a l ., 2012) , I he r e i n a na l y z e d A t g9 t r a f f i c ki ng unde r nor m a l a nd
l os s of U V R A G c ondi t i ons . T he l os s of U V R A G i nhi bi t e d A t g9 t r a ns l oc a t i on upon
a ut opha gy i nduc t i on ( f i gur e 1) , s ugge s t i ng a n i m por t a nt r ol e of U V R A G i n A t g9
t r a ns por t .
U V R A G c ont a i ns di f f e r e nt f unc t i ona l dom a i ns t o a s s oc i a t e w i t h di s t i nc t pa r t ne r s t o
c onduc t di f f e r e nt pa t hw a y s ( S . H e e t a l ., 2013) . I n t hi s s t udy , t hr e e f unc t i ona l dom a i ns ,
i nc l udi ng C 2 dom a i n, C C D dom a i n a nd 270- 442 dom a i n, w e r e a na l y z e d by
c om pl e m e nt a t i on a s s a y . M a t c hi ng w i t h de m ons t r a t i on t ha t U V R A G i s i nvol ve d i n A t g9
t r a ns por t upon a ut opha gy i nduc t i on, t he knoc kdow n of U V R A G di s r upt e d A t g9
t r a ns l oc a t i on a nd c om pl e m e nt a t i on of U V R A G w i l d t y p e t o e ndoge nous l e ve l ( f i gur e 2c )
on U V R A G - de pl e t e d c e l l s r e ve r s e d A t g9 di s pe r s i on. ( F i gur e 2a , t hi r d r ow ) I n c ont r a s t t o
27
w i l d- t y p e U V R A G c om pl e m e nt a t i on, C om pl e m e nt a t i on of U V R A G K 78A / R 82A m ut a nt
a nd U V R A G ∆ C C D m ut a nt di dn ’ t r e s t or e A t g9 t r a ns l oc a t i on a f t e r a ut opha gy i nduc t i on,
s ugge s t i ng t ha t C C D dom a i n of U V R A G i s r e qui r e d a nd P I 3P bi ndi ng i s r e qui r e d f or
A t g9 t r a ns l oc a t i on upon a ut opha gy i nduc t i on. H ow e ve r , our pr e vi ous s t udy ha s s how n
P I 3P - bound U V R A G m e di a t e s G ol gi - E R r e t r ogr a de t r a ns por t s o a s t o m a i nt a i n t he
s t a bi l i t y of G ol gi a nd E R s t r uc t ur e . B r e a ki ng t he bi ndi ng be t w e e n P I 3P a nd U V R A G c a n
r e s ul t c om pr om i s e d f unc t i ons of G ol gi a nd E R , a nd G ol gi a nd E R a r e a l s o t he s our c e of
A ut opha gos om e . T he r e f or e , i t i s pos s i bl e t he c om pr om i s e d f unc t i on of G ol gi a nd E R
m a i nl y l e a ds t o t he di s r upt i on of A T G 9 t r a f f i c ki ng. B ut t hi s pos s i bi l i t y i s de ni e d by t he
f a c t s t ha t c om pl e m e nt a t i on of U V R A G ∆ 270- 442 r e ve r s e d A t g9 t r a ns l oc a t i on ( F i gur e 2a ,
f or t h r ow ) a s w e l l a s t he knoc kdow n of R I N T 1 di dn ’ t i nhi bi t A t g9 di s pe r s i on a f t e r
a ut opha gy i nduc t i on ( F i gur e 3a , f or t h r ow ) . B e c a us e w he n R I N T 1, a pa r t of U V R A G -
P I 3 P - E R t e t he r c om pl e x, i s knoc ke d dow n, t he G ol gi a nd E R s t r uc t ur e c a n be di s r upt e d ( S .
H e e t a l ., 2013) , but t he r e i s no obs e r va bl e e f f e c t on A t g9 t r a f f i c ki ng.
C om bi ni ng c om pl e m e nt a t i on a s s a y w i t h t he B e c l i n1 knoc kdow n r e s ul t w hi c h r e ve a l e d
t he i nhi bi t i on of A t g9 t r a ns l oc a t i on ( F i gur e 3a ) , w e c a n c onc l ude t ha t t he i nt e r a c t i on
be t w e e n U V R A G a nd B e c l i n1 i s r e qui r e d f or A t g9 t r a f f i c ki ng a nd B e c l i n1 pr ot e i n i s a l s o
i nvol ve d i n A t g9 t r a ns por t upon a ut opha gy i nduc t i on.
G e ne r a l l y , t he A t g9 t r a f f i c ki ng i s f unc t i ona l l y r e l a t e d t o a ut opha gos om e bi oge ne s i s . T he
U V R A G - m e di a t e d A t g9 t r a f f i c ki ng c a n a l s o a f f e c t a ut opha gos om e bi oge ne s i s , ba s e d on
t he i m m unof l uor e s c e nt a s s a y , w hi c h de t e c t e d e ndoge nous A t g9 a nd L C 3 on U V R A G ,
B e c l i n1 a nd R I N T 1 knoc kdow n c e l l s . A f t e r i nduc t i on of a ut opha gy , de pl e t i on of
U V R A G i nt e r r upt e d A t g9 t r a ns l oc a t i on a nd r e s ul t e d i n a r e duc t i on of L C 3 punc t a t e
28
s t r uc t ur e . S i m i l a r l y , de pl e t i on of B e c l i n1 i nhi bi t e d A t g9 di s pe r s i on a nd ha d a de c r e a s e d
num be r of L C 3 s t r uc t ur e s c om pa r e d w i t h knoc kdow n c ont r ol gr oup. C onj uga t e d L C 3 i s
t he i ndi c a t or of a ut opha gos om e . W i t hout U V R A G or B e c l i n1, A t g9 c a nnot nor m a l l y
s uppl y m e m br a ne t o a ut opha gos om e s o t ha t t he f or m a t i on of a ut opha gos om e de c r e a s e s .
I a l s o ha ve e xa m i ne d t he ove r a l l a ut opha gy l e ve l s i n t he U V R A G ( W T a nd m ut a nt s ) -
e xpr e s s i ng c e l l s by i m m unobl ot t i ng w i t h a nt i bodi e s a ga i ns t p62 ( p62 de gr a da t i on i s a
r obus t m a r ke r of a ut opha gi c f l ux) ( N a ka i e t a l ., 2007) a nd L C 3 ( t he L C 3- I I l e ve l s
c or r e l a t e w i t h t he num be r of a ut opha gos om e s ) .
C ons i s t e nt l y , t he pr oduc t i on of L C 3- I I w a s gr e a t l y i nc r e a s e d i n t he W T - a nd Δ 270- 442-
e xpr e s s i ng c e l l s c om pa r e d w i t h t ha t i n t he Δ C C D - or K 78A / R 82A - e xpr e s s i ng c e l l s ( F i g.
5) . T o ve r i f y t ha t t he e nha nc e d l e ve l s of L C 3- I I r e pr e s e nt e d i nc r e a s e d a ut opha gy r a t he r
t ha n a n i m pa i r e d a ut opha gi c de gr a da t i on, I a s s a y e d t he l e ve l s of p62, a n a ut opha gi c
s ubs t r a t e . A s e x pe c t e d , W T a nd Δ 270 - 4 42 e x pr e s s i on m a r ke d l y r e duc e d t he l e ve l s of p62 ,
c om pa r e d w i t h w ha t w a s s e e n w i t h ve c t or , Δ C C D or K 78A / R 82A . T hus , U V R A G
m e di a t e d A t g9 t r a f f i c ki ng w oul d a f f e c t a ut opha gi c a c t i vi t y .
29
5 R e f e r e n c e s
1 . B r i n d a R a v i k u m a r , S h o u q i n g L u o , D u n e c a n C . O . M a s s e y , F i o n a M . M e n z i e s , K e v i n M o r e a u , U s h a
N a r a y a n a n , . . . M a i k e L i c h t e n b e r g . ( 2 0 1 0 ) . R e g u l a t i o n o f m a m m a l i a n a u t o p h a g y i n p h y s i o l o g y a n d
p a t h o p h y s i o l o g y . P h y s i o l o g i c a l R e v i e w s , 9 0 ( 4 ) , 1 3 8 3 - 1 4 3 5 . d o i : 1 0 . 1 1 5 2 / p h y s r e v . 0 0 0 3 0 . 2 0 0 9
2 . C u e r v o , A . M . , & W o n g , E . ( 2 0 1 0 ) . A u t o p h a g y g o n e a w r y i n n e u r o d e g e n e r a t i v e d i s e a s e s . N a t u r e
N e u r o s c i e n c e , 1 3 , 8 0 5 + . R e t r i e v e d f r o m
h t t p : / / g o . g a l e g r o u p . c o m / p s / i . d o ? i d = G A L E % 7 C A 2 3 0 4 8 3 3 3 0 & v = 2 . 1 & u = u s o c a l _ m a i n & i t = r & p = A O N E
& s w = w & a s i d = 3 c 8 0 5 3 7 c 6 0 8 2 4 2 5 0 4 c 2 1 c 0 1 5 e d c 8 0 2 c f
3 . G e n g , J . , & K l i o n s k y , D . J . ( 2 0 0 8 ) . T h e A t g 8 a n d A t g 1 2 u b i q u i t i n - l i k e c o n j u g a t i o n s y s t e m s i n
m a c r o a u t o p h a g y . E M B O R e p o r t s , 9 ( 9 ) , 8 5 9 - 8 6 4 . d o i : 1 0 . 1 0 3 8 / e m b o r . 2 0 0 8 . 1 6 3
4 . H e , C . , & L e v i n e , B . ( 2 0 1 0 ) . T h e b e c l i n 1 i n t e r a c t o m e . C u r r e n t O p i n i o n i n C e l l B i o l o g y , 2 2 ( 2 ) , 1 4 0 - 1 4 9 .
d o i : 1 0 . 1 0 1 6 / j . c e b . 2 0 1 0 . 0 1 . 0 0 1
5 . H e , S . , L i , B . , O h , S . , L e e , W . , T a k a h a s h i , Y . , W a n g , H . , . . . B h a r a t h a m , N . ( 2 0 1 3 ) . P t d I n s ( 3 ) P - b o u n d
U V R A G c o o r d i n a t e s g o l g i - E R r e t r o g r a d e a n d A t g 9 t r a n s p o r t b y d i f f e r e n t i a l i n t e r a c t i o n s w i t h t h e E R
t e t h e r a n d t h e b e c l i n 1 c o m p l e x . N a t u r e C e l l B i o l o g y , 1 5 ( 1 0 ) , 1 2 0 6 - U 1 9 2 . d o i : 1 0 . 1 0 3 8 / n c b 2 8 4 8
6 . H e , S . , O ' C o n n e l l , D . , Z h a n g , X . , Y a n g , Y . , & L i a n g , C . ( 2 0 1 4 ) . T h e i n t e r s e c t i o n o f g o l g i - E R r e t r o g r a d e
a n d a u t o p h a g i c t r a f f i c k i n g . A u t o p h a g y , 1 0 ( 1 ) , 1 8 0 - 1 8 1 . d o i : 1 0 . 4 1 6 1 / a u t o . 2 6 9 1 7
7 . I t a k u r a , E . , K i s h i , C . , I n o u e , K . , & M i z u s h i m a , N . ( 2 0 0 8 ) . B e c l i n 1 f o r m s t w o d i s t i n c t
p h o s p h a t i d y l i n o s i t o l 3 - k i n a s e c o m p l e x e s w i t h m a m m a l i a n A t g 1 4 a n d U V R A G . M o l e c u l a r B i o l o g y o f
t h e C e l l , 1 9 ( 1 2 ) , 5 3 6 0 - 5 3 7 2 . d o i : 1 0 . 1 0 9 1 / m b c . E 0 8 - 0 1 - 0 0 8 0
8 . K u m a , A . , H a t a n o , M . , M a t s u i , M . , Y a m a m o t o , A . , N a k a y a , H . , Y o s h i m o r i , T . , . . . M i z u s h i m a , N .
( 2 0 0 4 ) . T h e r o l e o f a u t o p h a g y d u r i n g t h e e a r l y n e o n a t a l s t a r v a t i o n p e r i o d . N a t u r e , 4 3 2 ( 7 0 2 0 ) , 1 0 3 2 -
1 0 3 6 . d o i : 1 0 . 1 0 3 8 / n a t u r e 0 3 0 2 9
9 . L a m b , C . A . , Y o s h i m o r i , T . , & T o o z e , S . A . ( 2 0 1 3 ) . T h e a u t o p h a g o s o m e : O r i g i n s u n k n o w n , b i o g e n e s i s
c o m p l e x . N a t u r e R e v i e w s . M o l e c u l a r C e l l B i o l o g y , 1 4 ( 1 2 ) , 7 5 9 - 7 7 4 . d o i : 1 0 . 1 0 3 8 / n r m 3 6 9 6
1 0 . L e v i n e , B . , & K r o e m e r , G . ( 2 0 0 8 ) . A u t o p h a g y i n t h e p a t h o g e n e s i s o f d i s e a s e . C e l l , 1 3 2 ( 1 ) , 2 7 - 4 2 .
d o i : 1 0 . 1 0 1 6 / j . c e l l . 2 0 0 7 . 1 2 . 0 1 8
30
1 1 . L i , L . , Z h a n g , X . , & L e , W . ( 2 0 1 0 ) . A u t o p h a g y d y s f u n c t i o n i n a l z h e i m e r ' s d i s e a s e . N e u r o d e g e n e r a t i v e
D i s e a s e s , 7 ( 4 ) , 2 6 5 - 2 7 1 . d o i : 1 0 . 1 1 5 9 / 0 0 0 2 7 6 7 1 0
1 2 . L i a n g , C . , A k a z a w a , C . , J u n g , J . U . , L e e , J . , I n n , K . , G a c k , M . U . , . . . F e n g , P . ( 2 0 0 8 ) . B e c l i n 1 - b i n d i n g
U V R A G t a r g e t s t h e c l a s s C v p s c o m p l e x t o c o o r d i n a t e a u t o p h a g o s o m e m a t u r a t i o n a n d e n d o c y t i c
t r a f f i c k i n g . N a t u r e C e l l B i o l o g y , 1 0 ( 7 ) , 7 7 6 - 7 8 7 . d o i : 1 0 . 1 0 3 8 / n c b 1 7 4 0
1 3 . L i a n g , C . , F e n g , P . , K u , B . , D o t a n , I . , C a n a a n i , D . , O h , B . , & J u n g , J . U . ( 2 0 0 6 ) . A u t o p h a g i c a n d
t u m o u r s u p p r e s s o r a c t i v i t y o f a n o v e l B e c l i n 1 - b i n d i n g p r o t e i n U V R A G . N a t u r e C e l l B i o l o g y , 8 ( 7 ) ,
6 8 8 - 6 9 8 . d o i : 1 0 . 1 0 3 8 / n c b 1 4 2 6
1 4 . L i a n g , C . , F e n g , P . , K u , B . , O h , B . , & J u n g , J . U . ( 2 0 0 7 ) . U V R A G : A n e w p l a y e r i n a u t o p h a g y a n d
t u m o r c e l l g r o w t h . A u t o p h a g y , 3 ( 1 ) , 6 9 - 7 1 . R e t r i e v e d f r o m
h t t p : / / w w w . l a n d e s b i o s c i e n c e . c o m / j o u r n a l s / a u t o p h a g y / a r t i c l e / 3 4 3 7 /
1 5 . L o z y , F . , & K a r a n t z a , V . ( 2 0 1 2 ) . A u t o p h a g y a n d c a n c e r c e l l m e t a b o l i s m . S e m i n a r s i n C e l l &
D e v e l o p m e n t a l B i o l o g y , 2 3 ( 4 ) , 3 9 5 - 4 0 1 . d o i : 1 0 . 1 0 1 6 / j . s e m c d b . 2 0 1 2 . 0 1 . 0 0 5
1 6 . M a r i , M . , G r i f f i t h , J . , R i e t e r , E . , K r i s h n a p p a , L . , K l i o n s k y , D . J . , & R e g g i o r i , F . ( 2 0 1 0 ) . A n A t g 9 -
c o n t a i n i n g c o m p a r t m e n t t h a t f u n c t i o n s i n t h e e a r l y s t e p s o f a u t o p h a g o s o m e b i o g e n e s i s . T h e J o u r n a l o f
C e l l B i o l o g y , 1 9 0 ( 6 ) , 1 0 0 5 - 1 0 2 2 . d o i : 1 0 . 1 0 8 3 / j c b . 2 0 0 9 1 2 0 8 9
1 7 . M a r t i n e z - V i c e n t e , M . , S u l z e r , D . , C u e r v o , A . M . , T a l l o c z y , Z . , W o n g , E . , T a n g , G . , . . . H a r r i s , S .
( 2 0 1 0 ) . C a r g o r e c o g n i t i o n f a i l u r e i s r e s p o n s i b l e f o r i n e f f i c i e n t a u t o p h a g y i n h u n t i n g t o n ' s d i s e a s e .
N a t u r e N e u r o s c i e n c e , 1 3 ( 5 ) , 5 6 7 - 5 7 6 . d o i : 1 0 . 1 0 3 8 / n n . 2 5 2 8
1 8 . M e l é n d e z , A . , & L e v i n e , B . ( 2 0 0 9 ) . A u t o p h a g y i n C . e l e g a n s . I n J a m e s M . K r a m e r a n d D o n a l d C .
M o e r m a n ( E d . ) , W o r m B o o k : T h e o n l i n e r e v i e w o f C . e l e g a n s b i o l o g y [ i n t e r n e t ] ( )
1 9 . M i z u s h i m a , N . ( 2 0 0 7 ) . A u t o p h a g y : P r o c e s s a n d f u n c t i o n . G e n e s & D e v e l o p m e n t , 2 1 ( 2 2 ) , 2 8 6 1 - 2 8 7 3 .
d o i : 1 0 . 1 1 0 1 / g a d . 1 5 9 9 2 0 7
2 0 . N a k a i , A . , A s a h i , M . , N i s h i d a , K . , H o r i , M . , M i z u s h i m a , N . , O t s u , K . , . . . M a t s u m u r a , Y . ( 2 0 0 7 ) . T h e
r o l e o f a u t o p h a g y i n c a r d i o m y o c y t e s i n t h e b a s a l s t a t e a n d i n r e s p o n s e t o h e m o d y n a m i c s t r e s s . N a t u r e
M e d i c i n e , 1 3 ( 5 ) , 6 1 9 - 6 2 4 . d o i : 1 0 . 1 0 3 8 / n m 1 5 7 4
31
2 1 . N e m c h e n k o , A . , C h i o n g , M . , T u r e r , A . , L a v a n d e r o , S . , & H i l l , J . A . ( 2 0 1 1 ) . A u t o p h a g y a s a t h e r a p e u t i c
t a r g e t i n c a r d i o v a s c u l a r d i s e a s e . J o u r n a l o f M o l e c u l a r a n d C e l l u l a r C a r d i o l o g y , 5 1 ( 4 ) , 5 8 4 - 5 9 3 .
d o i : 1 0 . 1 0 1 6 / j . y j m c c . 2 0 1 1 . 0 6 . 0 1 0
2 2 . O r s i , A . , R a z i , M . , D o o l e y , H . C . , R o b i n s o n , D . , W e s t o n , A . E . , C o l l i n s o n , L . M . , & T o o z e , S . A .
( 2 0 1 2 ) . D y n a m i c a n d t r a n s i e n t i n t e r a c t i o n s o f A t g 9 w i t h a u t o p h a g o s o m e s , b u t n o t m e m b r a n e
i n t e g r a t i o n , a r e r e q u i r e d f o r a u t o p h a g y . M o l e c u l a r B i o l o g y o f t h e C e l l , 2 3 ( 1 0 ) , 1 8 6 0 - 1 8 7 3 .
d o i : 1 0 . 1 0 9 1 / m b c . E 1 1 - 0 9 - 0 7 4 6
2 3 . R u s s e l l , R . C . , G u a n , K . , T i a n , Y . , Y u a n , H . , P a r k , H . W . , C h a n g , Y . , . . . D i l l i n , A . ( 2 0 1 3 ) . U L K 1
i n d u c e s a u t o p h a g y b y p h o s p h o r y l a t i n g b e c l i n - 1 a n d a c t i v a t i n g V P S 3 4 l i p i d k i n a s e . N a t u r e C e l l
B i o l o g y , 1 5 ( 7 ) , 7 4 1 - 7 5 0 . d o i : 1 0 . 1 0 3 8 / n c b 2 7 5 7
2 4 . S a i t o h , T . , & A k i r a , S . ( 2 0 1 0 ) . R e g u l a t i o n o f i n n a t e i m m u n e r e s p o n s e s b y a u t o p h a g y - r e l a t e d p r o t e i n s .
T h e J o u r n a l o f C e l l B i o l o g y , 1 8 9 ( 6 ) , 9 2 5 - 9 3 5 . d o i : 1 0 . 1 0 8 3 / j c b . 2 0 1 0 0 2 0 2 1
2 5 . S á n c h e z - P é r e z , A . M . , C l a r a m o n t e - C l a u s e l l , B . , S á n c h e z - A n d r é s , J . V . , & H e r r e r o , M . T . ( 2 0 1 2 ) .
P a r k i n s o n ' s d i s e a s e a n d a u t o p h a g y . P a r k i n s o n ' s D i s e a s e , 2 0 1 2 , 4 2 9 5 2 4 . d o i : 1 0 . 1 1 5 5 / 2 0 1 2 / 4 2 9 5 2 4
2 6 . S u z u k i , K . , K i r i s a k o , T . , K a m a d a , Y . , M i z u s h i m a , N . , N o d a , T . , & O h s u m i , Y . ( 2 0 0 1 ) . T h e p r e -
a u t o p h a g o s o m a l s t r u c t u r e o r g a n i z e d b y c o n c e r t e d f u n c t i o n s o f A P G g e n e s i s e s s e n t i a l f o r
a u t o p h a g o s o m e f o r m a t i o n . T h e E M B O J o u r n a l , 2 0 ( 2 1 ) , 5 9 7 1 - 5 9 8 1 . d o i : 1 0 . 1 0 9 3 / e m b o j / 2 0 . 2 1 . 5 9 7 1
2 7 . T a k a h a s h i , Y . , M e y e r k o r d , C . L . , H o r i , T . , R u n k l e , K . , F o x , T . E . , K e s t e r , M . , . . . W a n g , H . ( 2 0 1 1 ) .
B i f - 1 r e g u l a t e s A t g 9 t r a f f i c k i n g b y m e d i a t i n g t h e f i s s i o n o f g o l g i m e m b r a n e s d u r i n g a u t o p h a g y .
A u t o p h a g y , 7 ( 1 ) , 6 1 - 7 3 . R e t r i e v e d f r o m
h t t p : / / w w w . l a n d e s b i o s c i e n c e . c o m / j o u r n a l s / a u t o p h a g y / a r t i c l e / 1 4 0 1 5 /
2 8 . V a l d o r , R . , & M a c i a n , F . ( 2 0 1 2 ) . A u t o p h a g y a n d t h e r e g u l a t i o n o f t h e i m m u n e r e s p o n s e .
P h a r m a c o l o g i c a l R e s e a r c h : T h e O f f i c i a l J o u r n a l o f t h e I t a l i a n P h a r m a c o l o g i c a l S o c i e t y , 6 6 ( 6 ) , 4 7 5 -
4 8 3 . d o i : 1 0 . 1 0 1 6 / j . p h r s . 2 0 1 2 . 1 0 . 0 0 3
2 9 . W e b b e r , J . L . , & T o o z e , S . A . ( 2 0 1 0 ) . N e w i n s i g h t s i n t o t h e f u n c t i o n o f A t g 9 . F E B S L e t t e r s , 5 8 4 ( 7 ) ,
1 3 1 9 - 1 3 2 6 . d o i : 1 0 . 1 0 1 6 / j . f e b s l e t . 2 0 1 0 . 0 1 . 0 2 0
3 0 . W e b b e r , J . L . , Y o u n g , A . R . J . , & T o o z e , S . A . ( 2 0 0 7 ) . A t g 9 t r a f f i c k i n g i n m a m m a l i a n c e l l s . A u t o p h a g y ,
3 ( 1 ) , 5 4 - 5 6 .
32
3 1 . W i r t h , M . , J o a c h i m , J . , & T o o z e , S . A . ( 2 0 1 3 ) . A u t o p h a g o s o m e f o r m a t i o n - - t h e r o l e o f U L K 1 a n d
B e c l i n 1 - P I 3 K C 3 c o m p l e x e s i n s e t t i n g t h e s t a g e . S e m i n a r s i n C a n c e r B i o l o g y , 2 3 ( 5 ) , 3 0 1 - 3 0 9 .
d o i : 1 0 . 1 0 1 6 / j . s e m c a n c e r . 2 0 1 3 . 0 5 . 0 0 7
3 2 . X i e , Z . , & K l i o n s k y , D . J . ( 2 0 0 7 ) . A u t o p h a g o s o m e f o r m a t i o n : C o r e m a c h i n e r y a n d a d a p t a t i o n s . N a t u r e
C e l l B i o l o g y , 9 ( 1 0 ) , 1 1 0 2 - 1 1 0 9 . d o i : 1 0 . 1 0 3 8 / n c b 1 0 0 7 - 1 1 0 2
3 3 . Y a m a m o t o , H . , K a k u t a , S . , W a t a n a b e , T . M . , K i t a m u r a , A . , S e k i t o , T . , K o n d o - K a k u t a , C . , . . . O h s u m i ,
Y . ( 2 0 1 2 ) . A t g 9 v e s i c l e s a r e a n i m p o r t a n t m e m b r a n e s o u r c e d u r i n g e a r l y s t e p s o f a u t o p h a g o s o m e
f o r m a t i o n . T h e J o u r n a l o f C e l l B i o l o g y , 1 9 8 ( 2 ) , 2 1 9 - 2 3 3 . d o i : 1 0 . 1 0 8 3 / j c b . 2 0 1 2 0 2 0 6 1
3 4 . Y i n , X . , Z h a o , Y . , L i v e s e y , K . M . , T a n g , D . , C a o , L . , K a n g , R . , . . . X i e , M . ( 2 0 1 1 ) . U V i r r a d i a t i o n
r e s i s t a n c e - a s s o c i a t e d g e n e s u p p r e s s e s a p o p t o s i s b y i n t e r f e r i n g w i t h B A X a c t i v a t i o n . E M B O R e p o r t s ,
1 2 ( 7 ) , 7 2 7 - 7 3 4 . d o i : 1 0 . 1 0 3 8 / e m b o r . 2 0 1 1 . 7 9
3 5 . Y o r i m i t s u , T . , & K l i o n s k y , D . J . ( 2 0 0 5 ) . A u t o p h a g y : M o l e c u l a r m a c h i n e r y f o r s e l f - e a t i n g . C e l l D e a t h
a n d D i f f e r e n t i a t i o n , 1 2 ( S 2 ) , 1 5 4 2 - 1 5 5 2 . d o i : 1 0 . 1 0 3 8 / s j . c d d . 4 4 0 1 7 6 5
3 6 . Y o u n g , A . R . J . , C h a n , E . Y . W . , H u , X . W . , K ö c h l , R . , C r a w s h a w , S . G . , H i g h , S . , . . . T o o z e , S . A .
( 2 0 0 6 ) . S t a r v a t i o n a n d U L K 1 - d e p e n d e n t c y c l i n g o f m a m m a l i a n A t g 9 b e t w e e n t h e T G N a n d
e n d o s o m e s . J o u r n a l o f C e l l S c i e n c e , 1 1 9 ( P t 1 8 ) , 3 8 8 8 - 3 9 0 0 . d o i : 1 0 . 1 2 4 2 / j c s . 0 3 1 7 2
3 7 . Z h a o , Z . , C o s t a n z o , V . , S t a r k , J . M . , L i a n g , C . , O h , S . , L i , D . , . . . G h o z a l l i , I . ( 2 0 1 2 ) . A d u a l r o l e f o r
U V R A G i n m a i n t a i n i n g c h r o m o s o m a l s t a b i l i t y i n d e p e n d e n t o f a u t o p h a g y . D e v e l o p m e n t a l C e l l , 2 2 ( 5 ) ,
1 0 0 1 - 1 0 1 6 . d o i : 1 0 . 1 0 1 6 / j . d e v c e l . 2 0 1 1 . 1 2 . 0 2 7

Abstract (if available)

Abstract Autophagy is a cellular process of self‐digestion to maintain homeostasis and avoid cell death. The normal functioning of autophagy is strongly linked to its molecular pathway, especially the formation of autophagosome. Undoubtedly, the formation of autophagosome relies on the continuous supply of membrane source. Atg9, as the only transmembrane autophagy protein, plays an important role in membrane delivery for autophagosome formation. Here, I identify UVRAG, a Beclin1‐binding autophagic protein, mediates Atg9 trafficking upon autophagy induction to assist the autophagosome biogenesis as well as increase cellular autophagic activity. Meanwhile, the interaction between UVRAG and Beclin1 also is required for the Atg9 trafficking upon autophagy induction. These findings suggest UVRAG acts as an important regulator of Atg9 transport to further promote autophagosome formation when autophagy is induced.

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

A truncating mutation in the autophagy gene UVRAG drives inflammation and tumorigenesis in mice

PDF

Comparative studies of the replication of African and Asian strains of Zika virus

PDF

UVRAG maintains genomic integrity

PDF

Beclin 2-mediated autophagic degradation of the pathogenic proteins in neurodegenerative diseases

PDF

Mechanism of ethanol-mediated increase of SARS-COV-2 entry through GRP78 induction

PDF

UVRAG protects cells from UV-induced DNA damage by regulating global genomic nucleotide excision repair pathway

PDF

The role of amphisomes and Rab11 protein in release of mature hepatitis B viral particles and analysis of the arginine-rich C-terminal domain of HBV precore protein

PDF

Selective innate immune activation by murine gamma-herpesvirus 68 (ɣHV68)

PDF

Molecular mechanism for the immune evasion of CD1d antigen presentation by herpes simplex virus-1 UL56 protein

PDF

FoxO1 suppresses Kaposi's sarcoma-associated herpesvirus lytic replication and controls viral latency

PDF

Characterization of three novel variants of the MAVS adaptor

PDF

The effects of autophagy on hepatitis C virus

PDF

Herpes Simplex virus-1 UL56 collaborates with Nedd4 E3 ubiquitin ligase to downregulate surface CD1d and facilitate immune evasion of NKT cell function

PDF

Effect of hepatitis C virus infection on signal transduction adaptor protein: TRAF6

PDF

Virus customization of host protein machinery for efficient propagation

PDF

WAVE1 is a novel mediator of HPV trafficking to the Golgi apparatus

PDF

Modulation of KIF3A function by herpes simplex virus-1 US3 kinase in viral immune evasion

PDF

SARS-CoV-2 suppression of CD1d expression and NKT cell function

PDF

SARS-CoV-2 interaction with the CD1d/NKT cell antigen presentation system

PDF

Comparative studies of immune evasion function for herpes simplex virus-1 UL56 and US3 genes

Asset Metadata

Creator Zhang, Tian (author)

Core Title UVRAG mediates Atg9 transport upon autophagy induction

School Keck School of Medicine

Degree Master of Science

Degree Program Molecular Microbiology and Immunology

Publication Date 08/06/2014

Defense Date 03/19/2014

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag Atg9,autophagosome,autophagy,Beclin1,OAI-PMH Harvest,UVRAG

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Liang, Chengyu (committee chair), Feng, Pinghui (committee member), Yuan, Weiming (committee member)

Creator Email tinachueng69@gmail.com,zhangtia@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-453471

Unique identifier UC11287813

Identifier etd-ZhangTian-2779.pdf (filename),usctheses-c3-453471 (legacy record id)

Legacy Identifier etd-ZhangTian-2779.pdf

Dmrecord 453471

Document Type Thesis

Format application/pdf (imt)

Rights Zhang, Tian

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA