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Interactions between vesicular stomatitis virus G protein and the cytoskeletal factor protein 4.1

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Interactions between vesicular stomatitis virus G protein and the cytoskeletal factor protein 4.1

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Content INTERACTIONS BETWEEN VESICULAR STOMATITIS VIRUS G PROTEIN
AND THE CYTOSKELETAL FACTOR PROTEIN 4.1
Copyright 2002
by
Edward Hyun You
A Thesis Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY & MOLECULAR BIOLOGY)
December 2002
Edward Hyun You
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UMI Number: 1414864
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UNIVERSITY OF SOUTHERN CALIFORNIA
THE GRADUATE SCHOOL
UNIVERSITY PARK
LOS ANGELES, CALIFORNIA 90089-1695
This thesis, written by
________ E d w ard H vun You__________________
under the direction o f h i s thesis committee, and
approved by all its members, has been presented to and
accepted by the Director o f Graduate and Professional
Programs, in partial fulfillment o f the requirements for the
degree o f
Director
Thesis Committee
\air
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Table of Contents
List of Tables and Figures iii
Abstract iv
Chapter 1 Introduction 1
Chapter 2 Yeast Two-Hybrid Assay 6
Chapter 3 Cell Surface Expression Study 12
Chapter 4 Cell Surface Biotinylation Study 16
Chapter 5 Confocal Microscopy Study 25
Chapter 6 Discussion 31
References 33
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List of Figures and Tables
Figure 1
Figure 2
Table 1
Figure 3
Table 2
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
Figure 10
Figure 11
Figure 12
Overview of retrovirus assembly and budding.
Possible mechanisms leading to the basolateral
expression of VSV G in polarized epithelial cells.
BLAST sequence search results for VSV G
interacting clones.
Alternative splicing patterns leading to the various
isoforms of protein 4.1.
Scanning alanine mutagenesis effects on VSV G-
protein 4. I f interaction in the yeast two-hybrid
assay.
Cell surface expression levels of VSV G.
Cell surface analysis of MuLV envelope
glycoprotein and the MuLV chimera (MLV-G tail)
containing the VSV G cytoplasmic tail.
Surface biotinylation of MDCK type II cells stably
expressing VSV G.
Surface biotinylation of transiently transduced
MDCK type I cells.
Surface biotinylation of transiently transduced
MDCK type II cells.
VSV G surface biotinylation comparison of
transiently transduced MDCK type I cells versus
MDCK cells infected with vesicular stomatitis virus.
Subcellular colocalization of VSV G and protein 4.1
in confluent MDCK cells.
Subcellular colocalization of VSV G and protein 4.1
in confluent MDCK type I cells.
Subcellular localization of VSV G and zonula
occludens-2 in polarized MDCK type I cells.
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Abstract
We have shown an interaction occurring between the carboxy-terminal
fragment of protein 4.1 and the cytoplasmic tail of VSV G. Protein 4.1 is a
cytoskeletal protein first isolated and characterized from erythrocytes. Its function
is to link the cellular cytoskeleton to the plasma membrane by binding components
of the cytoskeleton to specific transmembrane proteins. By utilizing the yeast two-
hybrid system coupled with scanning alanine mutagenesis within the VSV G
cytoplasmic tail, we were able to identify key amino acids that may be crucial for
its interaction with protein 4.1. Some of the amino acids comprise a tyrosine-
based signaling motif that has been determined to be a membrane protein
internalization signal and a basolateral targeting signal. We have characterized
both the interaction between protein 4.1 and VSV G in 293T cells and the
functional significance of this interaction in the polarized Madin Darby canine
kidney cell system.
IV
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Chapter 1: Introduction
Many enveloped viruses assemble at the plasma membrane of their host cells.
The generally accepted model proposes that the envelope glycoproteins are initially
synthesized in the endoplasmic reticulum, modified in the Golgi network, and finally
transported to the plasma membrane as an oligomer (Fig. 1). The glycoproteins then
aggregate forming a “patch” on the surface whereupon the core proteins coalesce
underneath in the cytoplasm. Further processing and maturation eventually leads to
the budding of a viral particle from the host cell. Study of the assembly process has
shown that an interaction between the envelope glycoprotein and the core structural
proteins occurs in some viruses (4, 12). Host cell factors may be involved in the
assembly process as evidenced by the fact that cellular proteins have been isolated
from mature viral particles (7).
To further elucidate the assembly process, this study attempted to identify
any interactions between the cytoplasmic domains of viral glycoproteins and any
host cell factors. An initial yeast two-hybrid screen isolated an interesting
interaction between the 29-amino acid cytoplasmic tail of the vesicular stomatitis
virus G fusion protein and the carboxy-terminal fragment of the cytoskeletal protein,
protein 4.1 (p4.1f). Further characterization of the interaction found that a tyrosine-
based signaling motif (Y-T-D-I) within the VSV G cytoplasmic tail appeared to be
critical for the interaction with p4.1f. This signaling motif is comprised of a tyrosine
residue, followed by two amino acid residues (in this case threonine and glutamate),
and ends with a bulky hydrophobic amino acid (YXXO).
1
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Gore proteins r n a gemma
pf0t©lft3
tranfttailBfi
-a r" transcription
Figure 1. Overview of retrovirus assembly and budding.
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YXXO motifs have previously been shown to function as coated pit
internalization signals, although there is controversy over whether this particular
sequence plays that role in the context of VSV G (10, 11). In addition, the tyrosine
and isoleucine residues themselves (Y19 and 122) are of interest because it has been
reported to be essential for the basolateral targeting of VSV G in polarized epithelial
cells (10). Polarized cells are characterized by distinct apical and basolateral cell
surface domains with different morphologies, protein and lipid composition and
functions (6). The two membrane domains are separated from each other by tight
junctions, which form under conditions of cell-cell contact. The maintenance of these
domains requires the continual sorting of both newly synthesized and internalized
plasma membrane proteins, and the specific targeting of a number of enzymes, cell
surface receptors and viral proteins to these membranes have been described.
Although the YXXO has been characterized as a basolateral targeting signal, the
mechanism whereby transmembrane proteins expressing this signal in their
cytoplasmic domains are delivered and expressed in the basolateral membrane
domain is still largely unknown. The interaction between the YXXO motif and the
cytoskeletal factor, protein 4.1, may play a dual role in the basolateral expression of
VSV G. If the YXXO signaling motif within the VSV G cytoplasmic tail is acting as
an internalization signal, an interaction with protein 4.1 should inhibit proper
association with the AP-2 subunit of the coated pit internalization endocytosis
complex and thereby prevent internalization (Fig. 2). Moreover, interaction with a
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cytoskeletal element such as protein 4.1 should stabilize the expression of VSV G in
the membrane by linking the transmembrane protein to the underlying cytoskeleton.
This study characterizes the novel finding of an interaction between VSV G
and protein 4.1 and also addresses the proposed mechanism for the basolateral
expression of VSV G in polarized epithelial cells.
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*
VSV-G
Apical surface
I I II I I I I I I I ! I I I I II I I I I I
..................... l l l l l I l l 1
AP-2
Basolateral surface
VSV-G
AP-2
4.1
'Cytoskeleton
Figure 2. Possible mechanisms leading to the basolateral expression of VSV G in
polarized epithelial cells. VSV G expressed on the apical surface, where protein 4.1
is not present, is free to associate with the AP-2 subunit of the clathrin-coated pit
endocytosis complex leading to internalization. However, when expressed on the
basolateral side, VSV G interacts with protein 4.1 which then sterically inhibits
association with the AP-2 subunit and anchors VSV G to the cytoskeleton. Both
processes leads to the differential expression of VSV G on the basolateral membrane
domain.
5
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Chapter 2: Yeast Two-Hybrid Assay
An initial study was performed looking at possible interactions between the
cytoplasmic tails of HIV, VSV G, and MLV and any cellular factors. A yeast two-
hybrid assay (Matchmaker system, Clontech, Palo Alto, CA.) was used in order to
screen for a possible cellular partner of the cytoplasmic tail of VSV G. The
cytoplasmic domains of the vesicular stomatitis virus G fusion protein (VSV G) and
the envelope glycoproteins of the human immunodeficiency virus type I (HIV) and
the Moloney murine leukemia virus (MuLV) were fused in frame to the GAL4 DNA
binding domain in plasmid pAS2-l and screened against a 17-day old mouse embryo
cDNA library fused in frame to the GAL4 transcriptional activation domain in
plasmid pACT2. Both the HIV and MuLV cytoplasmic tail fusion proteins were
negative for any interaction. However, the 29 amino acid cytoplasmic region of
VSV G came up positive. Four million independent clones were screened for a
positive interaction, from which 6 different positive clones were obtained. The
cDNA sequences present in these clones were analyzed by performing BLAST
homology searches against protein and DNA sequence databases. The results are
shown below in Table 1.
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homology
clone #1 IL12
clone #3 carboxypeptidase
clone #13 protein 4.1
clone #19 no known homology
clone #25 pyruvate kinase
clone #29 epithelial protein for translation regulation
Table 1: BLAST sequence search results for VSV G interacting clones.
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The extent of the protein 4.1 sequences present in Clone #13 are shown in Figure 3.
This C-terminal fragment (p4.If) contains 8 exons , including 5 which are conserved
amongst all the isoforms of the protein 4.1 family (Fig. 3). Only exon 17 in the
putative spectrin/actin binding domain is present, but such an isoform has previously
been reported to exist in reticulocytes (2).
I wished to identify the sequence in the VSV G cytoplasmic tail responsible
for the interaction with p4.1f. This sequence contains two noteworthy motifs: a
polybasic region (KKR) and a YXXI motif.
Accordingly, I performed scanning alanine mutagenesis on selected residues
in the tail and screened the mutants for their ability to interact with p4.1f in the yeast
two-hybrid assay. I chose in particular to concentrate on residues that had previously
been analyzed for their contribution to basolateral targeting of an HA/VS V G
cytoplasmic tail chimeric protein in polarized MDCK cells (8), so that I could
directly compare the effects of the mutations on interactions in the yeast two-hybrid
system with the reported effects on basolateral targeting. These results implicated
the YXXI motif in particular in the p4. I f interaction, as mutation of both Y19 and
122 abolished the yeast two-hybrid interaction (Table 2). The reported observations
that these mutations also abolished basolateral targeting in an HA/VSV G chimera
(8) suggest the possibility that an interaction with protein 4.1 could underlie the
observed basolateral targeting and/or internalization of VSV G, as discussed below.
However, it was noted that other residues also abolished the yeast two-hybrid
interaction without affecting the reported basolateral distribution of the HA/VS V G
8
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Spectrin/A ctin
binding dom ain
UGA
.AUG
erythroid protein 4.1
12 13 17 18 19 20 21 22
clone#13 N '{ F \ v n f f W =^ c
D Constitutively expressed coding exons
I Alternatively spliced coding exons
IS U ntranslated regions
Figure 3. Alternative splicing patterns leading to the various isoforms of protein 4.1.
Specific isoform expression is dependent on cell type and differentiation stage of the
cell.
9
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chimera. These included residues on either side of the YXXI motif, including 118,
E23 and M24. In addition, it was also noted that while the individual substitutions of
residues K15 and R16 in the polybasic region gave good correlation between the
yeast results and the basolateral distribution analyses, the triple substitution of K14,
K15 and R16 with alanines had a greater effect in the yeast system. It is possible to
reconcile these discrepancies if it is considered that the yeast two-hybrid interaction
is inherently more sensitive than the basolateral targeting assay. Alternatively,
factors other than a putative interaction with protein 4.1 could be involved in
ensuring the basolateral targeting and/or maintenance of VSV G. A final caveat is
that in both of these assay systems, the cytoplasmic tail of VSV G is not being
assayed in its native form but as a fusion protein, which could also influence the
results.
10
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Phenotype Yeast Two-Hybrid Result Basolateral Membrane Expression*
Wild Typo
+ + + + + +
T13A
+ + + + + +
K15A + + +
R16A
+ +++
118 A -
+ + + +
Y19A - +
T20A + + +++
D21A
+ ++++
I22A - +
E23A -
+ + + +
M24A - + + +
Table 2. Scanning alanine mutagenesis effects on VSV G-protein 4. If interaction in
the yeast two-hybrid assay. Effects the mutations within the VSV G cytoplasmic tail
have on protein 4. If interaction are directly compared to the results similar mutations
had on VSV G basolateral distribution (Thomas et al., 1994).
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Chapter 3: Cell Surface Expression Study
Previous studies have established that the YXXO motif acts as an
endocytosis signal. This sequence of amino acids interacts with the AP-2 subunit of
the clathrin-coated pit mediated endocytosis mechanism. As a result, transmembrane
proteins expressing this signaling motif are subject to internalization and subsequent
degradation or intracellular transport. Disruption of this signaling motif decreases
internalization and increases the cell surface expression levels of the transmembrane
protein.
In order to test the hypothesis that the YXXO motif in the VSV G
cytoplasmic tail acts as an endocytosis signal, VSV G cell surface expression levels
were measured by fluorescence-activated cell sorting (FACS) analysis of 293T cells
transiently transfected with either the wild-type or Y19A mutant of VSV G, with and
without p4.1f. The 293T cells were transfected with 2.0 |_ ig of the VSV G expression
vector pCG and 5.0 pg of either the p4.1f expression plasmid pSA90-p4.1f, or the
empty vector pSA90, in order to keep the total amount of DNA in the transfections
constant. Forty-eight hours later, the cells were washed 3 times in PBS containing
10% goat serum (Invitrogen, Carlsbad, CA.). Cells were then incubated with a
polyclonal rabbit anti-VSV antibody (Bethyl Labs, Montgomery, TX.) at a dilution
of 1:400 in PBS-goat serum at 4°C for 1 hr. Next the cells were washed three times
with PBS-goat serum. For labeling, the 293T cells were incubated with a
corresponding goat anti-rabbit FITC-conjugated secondary antibody at a dilution of
12
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1:1000 (Pierce, Rockford, IL.) for 1 hr at 4°C. The cells were again washed three
times with PBS-goat serum, resuspended and fixed in 4% paraformaldehyde-PBS.
Labeled cells were then analyzed in a Coulter Epics XL FACS scanner (Beckman-
Coulter, Miami, FL).
The results (Fig. 4) demonstrated that the level of cell surface VSV G protein
was indeed enhanced by the Y19A mutation. Furthermore, although the cell surface
expression of the wild-type VSV G was increased by the co-expression of the protein
4.1 fragment, this enhancement was not seen in the cells expressing the Y19A
mutant.
In addition, as a control, the ecotropic Moloney leukemia virus (MuLV)
envelope protein was also used. The increase in cell surface expression appeared to
be VSV G-specific, as the ecotropic MuLV envelope glycoprotein was not affected
by the presence of the protein 4.1 fragment, despite the presence in the cytoplasmic
tail of the YXXO motif. A chimeric MuLV envelope protein was constructed where
its cytoplasmic tail domain was replaced with the VSV G cytoplasmic tail sequence
(MLV-Gt). When this chimeric construct was co-expressed with the protein 4.1
fragment, MLV-Gt surface expression was increased (Fig. 5).
This data supports the model that the YXXO signal motif in the VSV G
cytoplasmic tail region is indeed acting as an internalization signal, and that an
interaction with protein 4.1 or mutation of the signal potentiates the cell surface half-
life of VSV G.
13
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0 >
o
c
d >
u
M
< 1 >
o
3
4 >
3
O
W
Si
<
300000
250000
200000
150000
50000
0
Protein 4.1 F:
- +
VSV G
+
VSV G Y19A
Figure 4. Cell surface expression levels of VSV G. Wild-type VSV G and the VSV
G mutant Y19A were expressed alone or with protein 4.If in transiently transfected
293T cells. Polyclonal anti-VSV antibody in conjunction with a FITC-conjugated
secondary antibody were used to label surface VSV G. Labeled cells were then
analyzed using fluorescence-activated cell sorting (FACS). Each data set is the
result of five replicate experiments. Error bars represent the standard deviation.
14
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a >
u
c
O
«
0 >
v-
O
3
_2
o
u
jQ
<
250000
200000
150000
100000
50000
Protein 4.1F: + +
MLV MLV-G tail
Figure 5. Cell surface analysis of MuLV envelope glycoprotein and the MuLV
chimera (MLV-G tail) containing the VSV G cytoplasmic tail. An antibody (83A25)
recognizing the SU ectodomain of the MuLV envelope glycoprotein was used to
probe the cell surface. A FITC-conjugated secondary antibody was used as before,
and cells were analyzed using FACS analysis. Each data set is the result of five
replicate experiments. Error bars represent the standard deviation.
15
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Chapter 4: Cell Surface Biotinylation Study
In order to study VSV G expression in the context of a polarized epithelial
cell system, Madin-Darby canine kidney cells (MDCK) were utilized. Both MDCK
cell types were acquired, type I (high resistance, CCL-34, ATCC, Manassas, VA.)
and type II (low resistance, courtesy of Dr. Curtis Okamoto, USC, Los Angeles,
CA.) Cells were transduced with a retroviral vector expressing VSV G.
Susceptibility to infection of MDCK cells by MuLV-based retroviral vectors
pseudotyped with VSV G was determined first. The vectors were produced by
transient transfection (calcium phosphate treatment) of a 50% confluent 6 cm plate
of 293T cells with 5pg each of three expression plasmids - plasmid pHIT60, which
expresses MuLV Gag-Pol, the retroviral vector genome pCnBg, which expresses the
P-galactosidase marker gene, and pCG, which is a CMV promoter driven expression
vector of VSV G. This system for the rapid generation of retroviral vectors is
described in more detail in Soneoka et al. 1995. The method reproducibly produces
high titer retroviral vectors (greater than 106 colony forming units (CFU) per ml) in
the supernatant of the 293T culture by 48 hrs. post-transfection.
MDCK cells were transduced with the VSV G pseudotyped retroviral vectors
by incubating a 50% confluent 6 cm plate of MDCK cells with 3 ml of filtered vector
supernatant. Forty-eight hours later, the cells were stained for P-galactosidase
activity in order to assess the efficiency of gene transfer. Both the type I and type II
MDCK cells were shown to be efficiently transduced by the retroviral vectors, with
>50% of the cells expressing P-galactosidase two days after transduction.
16
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In order to establish a cell population stably expressing VSV G, MDCK cells
were transduced with pHP-VSV G vector stocks, generated by the transfection of
293T cells with 5 pg each of pHIT60 and pHP-VSV G. Vector pHP-VSV is a
retroviral vector expressing VSV G from the retroviral LTR promoter and a
puromycin resistance gene from an internal SV40 promoter. The vectors so produced
were used to transduce sub-confluent MDCK cells, followed by selection with 2.5
pg/ml puromycin for 3 days. VSV G expression was confirmed by Western analysis
(data not shown).
MDCK cells were also infected with wild-type VSV (Indiana strain) to
promote VSV G expression. Confluent MDCK monolayers were infected with VSV
at a concentration of 0.9 pfu/'ml. Experiments were performed 3 hours post-infection
since prolonged infection led to the disruption and loss of integrity of the MDCK
monolayer. The resulting MDCK/VSV G cells were plated onto 0.4 pm transwell
filters (Costar) and allowed to become polarized over several days. The degree of
polarization was determined by measuring the resistance (Q) across the cellular
monolayer and calculating the transepithelial resistance (Q x cm2). Based on
previous studies of MDCK cells, a transepithelial resistance greater than 1000
Q x cm was taken as evidence for cellular polarization for the MDCK type I cells
and >500 Q x cm2 for type II cells.
For surface quantification of VSV G, surface proteins were labeled with
sulfo-NHS-SS-biotin (Pierce, Rockford, 1 1 1 .) (50 mg/ml in ice-cold PBS containing
17
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Mg2 + and Ca2 + ). To differentiate between the two membrane surfaces, 1.5 ml of the
labeling mix was added to the basolateral surface and 0.5 ml on the apical surface.
Filters were then incubated on ice for 30 min with very gently horizontal rocking
motion to ensure mixing. The biotin solution was removed, and any excess biotin
was quenched in two ice-cold washes of PBS-Mg-Ca containing 100 mM glycine.
Transwell filters were then excised from the filter cup with a razor blade. The
biotinylated cells were then lysed on ice in 1ml of lysis buffer (1.0% Triton X-100,
150 mM NaCl, 5 mM EDTA, 50 mM Tris, pH 7.5) for 1 hr. Cell lysates were then
scraped off the filter and clarified by centrifugation at 14,000 x g for 10 min at 4°C.
The upper 900 ul supernatant phase was collected and placed into a separate
microcentrifuge tube containing 100 ul of packed streptavidin-agarose beads (Pierce,
Rockford, 11 1 .). Lysates were incubated overnight (12-16 hr) at 4°C with end-over-
end rotation. Supernatants were then aspirated off the beads. Beads were washed
three times with lysis buffer, twice with high-salt buffer (same as lysis buffer except
0.1% Triton X-100), and once with no-salt wash buffer (10 mM Tris, pH 7.5).
Proteins were eluted from beads by adding 30 ul SDS-containing sample buffer (80
mM DL-dithiothreitol, 5.0% SDS, 0.008% bromophenol blue, 0.24 M Tris-HCl, pH
8.0) and heated at 95°C for 5 min. Samples were loaded onto a 4-12% gradient Tris-
Glycine gel (Invitrogen, Carlsbad, CA.). For detection of the VSV G protein, blots
were probed with a rabbit polyclonal anti-VSV antibody (Bethyl Labs, Montgomery,
TX.) at 1:5,000 dilution. The secondary antibody used was a horseradish
peroxidase-conjugated goat anti-rabbit immunoglobulin (Ig) G at a dilution of
18
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1:10,000 (Pierce, Rockford, 111.). Specific protein signals were detected using an
enhanced chemiluminsecence (ECL) kit (Amersham, Arlington Heights, IL.).
Previous studies of VSV G distribution in polarized MDCK cells
characterized the protein to be predominantly expressed on the basolateral domain
(1,10,11). However, up until recently, most of the data had been derived using
electron microscopy techniques, or chimeric proteins containing only portions of
VSV G were used rather than the wild-type protein. Biotinylation followed by
protein quantitation of VSV G provides a more precise method of characterizing the
protein within the MDCK membrane domains.
Initially, MDCK cells were transduced with MuLV vectors containing the
VSV G gene. Subsequent selection in puromycin yielded a stable population
expressing VSV G at high levels. However, biotinylation results differed widely
from published data. There was a greatly enhanced apical expression (-90% of the
signal found on the apical surface) of the VSV G Y19A mutant which is to be
expected since the basolateral targeting signal is disrupted. This follows the
generally accepted dogma. But, wild-type VSV G displayed an expression pattern
similar to that of the Y19A mutant (Fig. 6). This occurred continually with each
stable MDCK (type I and type II) population that was produced. It was proposed
that, although the MDCK cells were stably expressing VSV G, the levels of protein
expression were higher than norm ally found physiologically. This m ay have
saturated the protein transport pathways regulating the differential distribution of
proteins containing the YXXO signal. To address this issue, MDCK cells were
transiently transduced to express VSV G. Cells were plated on Transwell filters at
19
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Lane
1
2
3
4
5
6
M D CK Apical
M D CK Basolateral
MDCK-VSVG Apical
MDCK-VSV G Basolateral
M D CK-VSV G Y19A Apical
M D CK-VSV G Y19A Basolateral
Figure 6. Surface biotinylation of MDCK type II cells stably expressing VSV G.
Two panels represent separate experiments where cell surface proteins were first
biotinylated, probed with a polyclonal anti-VSV antibody and then probed with a
horse radish peroxidase-conjugated secondary antibody. No real difference can be
seen in the apical/basolateral distribution between the wild-type VSV G and the VSV
G Y19A mutant.
20
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higher density and transduced with the MuLV vectors. Cells were placed under
puromycin selection only until complete monolayer formation occurred
(2-3 days versus 5-7 days of stable selection). Polarization was verified by
monitoring the transpeithelial resistance (>1000 Q x cm2 for type I cells; >500 Q x
cm2 for type II cells). Under these conditions, wild-type VSV G was predominantly
on the basolateral surface (-70%) albeit not as high as it has been reported in the
literature (-90%) in the MDCK type I cells. The VSV G Y19A mutant again
displayed almost total apical expression (Fig. 7). The MDCK type II cells still
displayed disparate basolateral distribution of VSV G either with transient
transduction or by infection with VSV (Fig. 8). Therefore, at this point, experiments
continued only utilizing the MDCK type I high-resistance cells.
To verify the data found using transient transduction, MDCK type I cells
were infected with wild-type VSV and immediately biotinylated. As shown in
Figure 9, transiently transduced VSV G-expressing MDCK type I cells display a
similar pattern when compared to VSV-infected cells.
This method of differential biotinylation has been optimized for quantifying
VSV G, the entire protein, in a polarized cell system. Moreover, transiently
transducing MDCK type I cells to express VSV G and the VSV G mutants allows for
characterization of the fusion protein in an epithelial cell system at an expression
level similar to that of a wild-type infection. The next step would be to characterize
the VSV G mutants first analyzed in the yeast two-hybrid system and monitor any
changes in the distribution of VSV G. The same experiments can be performed
while co-expressing protein 4. If and overexpressing wild-type protein 4.1.
21
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MDCK MDCK-VSV G MDCK-VSV G
Y15A
Ap Baso Ap Baso Ap Baso
A.
B.
C.
Figure 7 . Surface biotinylation of transiently transduced MDCK type I cells [A.].
MDCK cells were transduced and placed under selection only until complete
monolayer formation and polarization. Polarization was checked by measuring
• 9 • •
transepithelial resistance (>1000 Q x cm ), checking for the basolateral expression of
biotinylated Na/K-ATPase [B.], and checking for the apical secretion of the secretory
protein, gp80 [C.]. Wild-type VSV G shows a predominant basolateral expression
pattern. VSV G Y 19A on the other hand shows an enhanced apical distribution.
VSV G
Na/K-ATPase
gp80
IllBl
■ 1 1 1
2 2
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MDCK MDCK-VSVG MDCK-VSVG MDCK
Y19A VSV(I)
Ap B aso Ap B aso Ap B aso Ap Baso
a y ' jM gn n g * * m k 4 V SV G
- .....' / ,. . . . ............. : ,mt&PIPf •-;“ '“ ! if'“'
Figure 8. Surface biotinylation of MDCK type II cells. MDCK type II cells were
transiently transduced with MuLV vectors to express VSV G constructs. Cells were
then placed under puromycin selection only until the formation of a complete
monolayer. Another set of MDCK type II cells were infected with wild-type
vesicular stomatitis virus (0.9 pfu/ml) on the Transwell filter. Subsequent
biotinylation shows an increased apical signal for the VSV G Y19A mutant;
however, wild-type VSV G also displays a higher apical signal both in the transiently
transduced and in the infected population.
23
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MDCK MDCK MDCK MDCK
VSV-G VSV-G Y1.9A VSV infected
Ap Bl Ap Bl Ap Bl Ap Bl
VSV-G
Figure 9. VSV G surface biotinylation comparison of transiently transduced MDCK
type I cells versus MDCK cells infected with vesicular stomatitis virus. Both the
transiently transduced and VSV-infected MDCK cells display a predominantly
basolateral VSV G signal.
24
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Chapter 5: Confocal Microscopy Study
To verify the interaction between VSV G and protein 4.1 and to locate their
site(s) of their interaction, double-label immunofluorescence confocal microscopy
was used to study the distribution of VSV G and protein 4.1 in confluent Madin-
Darby canine kidney (MDCK) epithelial type I cells stably expressing VSV G.
MDCK cells grown on 0.4 pm Transwell filters (Corning-Costar, Cambridge, MA.)
to 50% confluency were first transduced using MuLV retroviral vectors that included
a transfer gene expressing VSV G and a puromycin-resistance gene cassette (pHP-
VSV G). Cells were then put under puromycin selection (2.5 mg/ml) until a
complete monolayer had formed. Polarization was determined by confirming a
transepithelial resistance >1,000 Q. x cm2. Monolayers were washed in PBS
2+ 2+
containing Mg and Ca . Cells were then fixed in 4% paraformaldehyde. Cultures
were then washed three times with PBS-Mg-Ca and incubated 15 min in PBS-Mg-Ca
containing 50 mM NH4CI. After washing, the cultures were permeabilized with 0.1%
Triton X-100 in PBS-Mg-Cafor 30 min and then incubated 2 h with the following
antibodies: mouse monoclonal anti-VSV G immunoglobulin (Ig) G (Sigma-Aldrich,
St. Louis, MO.) and rabbit polyclonal anti-protein 4.1 [anti-20] (synthesized
antibody whose epitope lies in exon 20 of protein 4.1) (Bethyl Laboratories,
Montgomery, TX.) or rabbit polyclonal anti-zonula occludens-2 [anti-ZO-2](Zymed,
San Francisco, CA.). Antibodies were suspended in PBS-Mg-Ca supplemented with
1% BSA and incubated on filters for 1 hr at a dilution of 1 GOO. After several
washes, the cultures were incubated 30 min with FITC-conjugated goat anti-mouse
25
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IgG (1:200 dilution; Sigma-Aldrich, St. Louis, MO.) and Cy3-conjugated goat anti
rabbit IgG (1:200 dilution; Sigma-Aldrich, St. Louis, MO.) in the same buffer. Filters
were washed with PBS-Mg-Ca, mounted in PBS/glycerol, and observed with a Zeiss
laser scanning microscope (LSM 510; Zeiss, Thornwood, NY.). As shown in Fig.
10, VSV G (shown in red, Fig 10A) and protein 4.1 (green, Fig 10B) localized at the
cell-cell contacts and displayed honeycomb-like staining patterns. By superimposing
Fig A and B, the yellow color (Fig 10C) produced due to the combination of the red
and green colors suggests the colocalization of VSV G and protein 4.1 at the cell-cell
junctions of confluent MDCK cells.
A more definitive analysis required a lateral scan of the epithelial monolayer;
however, due to bleaching effects of the FITC and Cy3 fluorophors under prolonged
laser exposure, scanning through the entire layer was impossible. At this point, a
different set of secondary antibodies which were conjugated to Alexa Fluor
fluorescent markers (Molecular Probes, Eugene, OR.) were used for confocal
scanning. These new secondary antibodies were able to maintain adequate
fluorescence under prolonged laser excitation allowing for scanning of images
through the epithelial monolayer. The experiment was repeated using monoclonal
Alexa Fluor 488-conjugated goat anti-rabbit antibody in lieu of the FITC-conjugated
secondary antibody, and Alexa Fluor 568-conjugated goat-anti-rabbit antibody was
substituted for the Cy3-conjugated secondary antibody. Antibody concentrations
and incubation times were all the same. In Fig. 11, VSV G (red, Fig. 11 A) and
protein 4.1 (green, Fig. 1 IB) were again scanned, and the resulting images were
26
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Figure 10. Subcellular colocalization of VSV G and protein 4.1 in confluent MDCK
type I cells. Confluent MDCK cells were fixed and processed for
immunofluorescence using antibodies for VSV G and protein 4.1 as described. VSV
G (A) and protein 4.1 (B) localize at cell-cell contacts in a continuous fashion. The
yellow color in C indicates the colocalization VSV G and protein 4.1.
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Figure 11. Subcellular colocalization of VSV G and protein 4.1 in confluent MDCK
type I cells. Confluent MDCK cells were fixed and processed for
immunofluorescence using antibodies for VSV G and protein 4.1. Alexa Fluor-
conjugated secondary antibodies were used. VSV G (A and D) and protein 4.1 (B
and E) again localize at cell-cell contacts in a continuous fashion. When the two
panels are overlayed, the yellow color in panels C and F indicate the colocalization
VSV G and protein 4.1. Panels D, E, and F are transverse sections. Note that the
transverse sections of VSV G (D) and protein 4.1 (E) indicate that both proteins
localize along the lateral membrane domain with a high concentration near the area
where tight junctions occur.
28
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overlayed to confirm the same colocalization patterns which appeared using the first
set of secondary antibodies (Fig. 11C). Moreover, lateral views show VSV G and
protein 4.1 distributing along the lateral membrane domain of the cells with some
concentration occurring high in the lateral domains where tight junctions are known
to occur. Protein 4.1 was previously characterized in the same cellular location in a
study characterizing an interaction between protein 4.1 and a component of the tight
junction, zonula occludens-2 (5).
As a control, MDCK cells were stained for VSV G (red, Fig. 12A) and
zonula occludens-2 (green, Fig. 12B). Staining patterns appeared to be similar both
in the overview and in the lateral view to the VSV G/protein 4.1 dual stains. This is
to be expected since it has already been established that protein 4.1 interacts with
zonula occludens-2 (5).
The confocal data presented does not provide direct evidence of a physical
interaction between protein 4.1 and VSV G; however, this study does show that the
two proteins have similar distributions within the cell. Moreover, the distribution
pattern of VSV G appears to corroborate the protein levels seen in the previous
biotinylation experiments. Interestingly, VSV G appears to show a more prevalent
“lateral” plasma membrane distribution compared to “basal”. This lateral expression
with a concentration near the tight junctions is the first to be described for the VSV
G fusion protein.
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E.
F.
Figure 12. Subcellular localization of VSV G and zonula occludens-2 in polarized
MDCK type I cells, VSV G (A and D) and zonula occludens-2 (B and E) localize at
the cell-cell junctions. Lateral views (D and E) show that both proteins display a
distinct lateral membrane distribution. Both show a concentration near the tight
junction region where zonula occludens-2 is a component. Overlays in panels C and
F show yellow indicating regions of colocalization.
30
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Chapter 6: Discussion
The vesicular stomatitis virus G fusion protein (VSV G) has been routinely
used as a model for differential transport and expression of proteins in polarized
epithelial cell systems. Although targeting signals regulating the basolateral
expression of VSV G have been identified, the mechanism is still not understood.
The discovery of a novel interaction between the cytoplasmic tail of VSV G and
protein 4.1 presented the possibility that the submembranous cytoskeleton of a
polarized cell may play a role in the basolateral expression of VSV G. I have
described two possiblities to account for a role of protein 4.1 in the establishment
and/or maintenance of the basolateral distribution of VSV G. Either protein 4.1
masks a putative internalization signal and therefore increases the half-life of VSV G
on the cell surface, or the interaction with protein 4.1 anchors VSV G to the
cytoskeleton. The cell surface expression experiments I described attempted to
elucidate the significance of this interaction and in particular to address the question
of whether an interaction between the two proteins functions to modulate the
exposure of an internalization signal within the VSV G cytoplasmic tail to the
endocytic machinery. The data shows that the YXXO motif within the VSV G
cytoplasmic tail does appear to act as an internalization signal. Co-expression of the
protein 4.1 fragment and mutation of the tyrosine residue both resulted in the
increase of VSV G surface levels which supports the model that an interaction with
protein 4.1 modulates the internalization signal. I have also described two methods
to specifically characterize the subcellular distribution of VSV G in polarized
MDCK cells (surface biotinylation and confocal microscopic analysis). Both
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experiments should be extended to study the degree of colocalization between VSV
G and the panel of VSV G mutants first characterized in the yeast two-hybrid with
protein 4.1 and the protein 4.1 fragment. These approaches will hopefully elucidate
a possible mechanism for the differential expression of viral proteins in polarized
cells. An increased understanding of the mechanisms whereby proteins are
selectively targeted and/or retained in discrete membrane domains has implications
for the field of protein transport and virus assembly.
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References
1. Brewer, C. and M.G. Roth. 1995. Polarized exocytosisin MDCK cells is
regulated by phosphorylation. J. Cell Sci. 106:789-796.
2. Conboy, J.G., Chan, J.Y., Chasis, J.A., Kan, Y.W., andN. Mohandas. 1991.
Tissue- and development-specific alternative RNA splicing regulates expression
of multiple isoforms of erythroid membrane protein 4.1. J. Biol. Chem.
266:8273-8280.
3. Le Maout, S., Brejon, M., Olsen, O., Merot, J., and Paul A. Welling. 1997.
Basolateral membrane targeting of a renal-epithelial inwardly recifying
postassium channel from the cortical collecting duct, CCD-IRK3, in MDCK cells.
Proc. Natl. Acad. Sci. USA. 94:13329-13334.
4. Lo, S.Y., Selby, M.J., and J.H. Ou. 1996. Interaction between hepatitis core
protein and El envelope protein. J. Virol. 70:5177-5182.
5. Mattagajasingh, S.N., Huang, S.C., Hartenstein, J.S., and E.J. Benz Jr. 2000.
Characterization of the interaction between protein 4.1R and ZO-2. A possible
link between the tight junction and the actin cytoskeleton. J. Biol. Chem.
275:30573-30585.
6. Matter, K. and I. Mellman. 1994. Mechanisms of cell polarity: sorting and
transport in epithelial cells. Curr. Opin. Cell Biol. 6:545-554.
7. Ott, D.E., Coren, L.V, Johnson, D.G., Kane, B.P., Sowder, R.C 2nd, Kim, Y.D.,
Fisher, R.J., Zhou, X.Z., Lu, K.P., and L.E. Anderson. 2000. Actin-binding
cellular proteins inside human immunodeficiency virus type 1. Virology 266:42-
51.
8. Soneoka, Y., Cannon, P.M., Ramsdale, E.E., Griffiths, J.C., Romano, G.,
Kingsman, S.M., and A.J. Kingsman. 1995 A transient three-plasmid expression
system for the production of high titre retroviral vectors. Nucl. Acid Res. 23:628-
633.
9. Thomas, D.C., and M.G. Roth. 1994. The basolateral targeting signal in the
cytoplasmic domain of glycoprotein G from vesicular stomatitis virus resembles a
variety of intracellular targeting motifs related by primary sequence but having
diverse targeting activities. J. Biol. Chem. 269:15732-15739.
10. Thomas, D.C., Brewer, C.B., and M.G. Roth. 1993. Vesicular stomatitis virus
glycoprotein contains a dominant cytoplasmic basolateral sorting signal critically
dependent upon a tyrosine. J. Biol. Chem. 268:3313-3320.
33
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11. Trowbridge, I.S., Collawn, J.F., and C.R. Hopkins. 1993. Signal-dependent
membrane protein trafficking in the endocytic pathway. Annu. Rev. Cell Biol.
9:129-161.
12. Wyma, D.J., Kotov, A., and C. Aiken. 2000. Evidence for a stable interaction of
gp41 with Pr55(Gag) in immature human immunodeficiency virus type 1
particles. J. Virol. 74:9381-9387.
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Asset Metadata

Creator You, Edward Hyun (author)

Core Title Interactions between vesicular stomatitis virus G protein and the cytoskeletal factor protein 4.1

School Graduate School

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

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

Tag biology, cell,chemistry, biochemistry,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Anderson, W. French (committee chair), [illegible] (committee member), Mircheff, Austin K. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-299916

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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 au...

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