UVRAG mediates Atg9 transport upon autophagy induction

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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 .,
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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 .
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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
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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’ .
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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
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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

Asset Metadata

Creator Zhang, Tian (author)

Core Title UVRAG mediates Atg9 transport upon autophagy induction

Contributor Electronically uploaded by the author (provenance)

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:digitallibrary.usc.edu:usctheses,OAI-PMH Harvest,UVRAG

Format application/pdf (imt)

Language English

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

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.