 |
INTRODUCTION |
The yeast Rvs161p and Rvs167p, together with mammalian amphiphysin
I and II, nematode, and fission yeast isoforms, constitute a family of
conserved proteins (1). The N termini of the different proteins share
the highest homology, and this common domain was called the BAR domain
(BIN/Amphiphysin/RVS domain (2)).
Rvs161p consists only of the BAR domain (Fig. 1), whereas the other
members of the family have an SH3 domain at their C termini and a
central domain varying among the different proteins. In the case of
Rvs167p, the central domain is rich in glycine, proline, and alanine
and therefore is called the GPA domain (Fig. 1).
The mammalian homolog, amphiphysin I, was first identified as a brain
protein enriched at presynaptic regions (3). The identification of
dynamin, synaptojanin, the
c-subunit of AP2-adaptin, and
clathrin as amphiphysin I-interacting proteins further implicated amphiphysin I in endocytosis (1). A more broadly expressed isoform,
amphiphysin II, has been identified by several groups and was found to
interact with the same proteins that interact with amphiphysin I (1).
Interestingly, the two isoforms can be coimmunoprecipitated from brain
extracts suggesting that they act in concert (4). Indeed, two studies
(4, 31) found that the two amphiphysin isoforms colocalize in brain.
However, a third study identified a different subcellular localization of the two isoforms (5). Amphiphysin I was shown to be concentrated in
the cortical cytoplasm of nerve terminals, whereas amphiphysin II was
concentrated in axon initial segments and nodes of Ranvier (5).
The two yeast members of the amphiphysin family, encoded by
RVS161 and RVS167, were first identified in a
screen for mutations causing reduced viability upon nutrient starvation
(6, 7). RVS161 was also identified in a screen for
endocytosis mutants (8). Mutations in rvs161 or
rvs167 exhibit the same phenotypes except for a defect in
cell fusion only found in the rvs161 mutant (9). The mutant
phenotypes include defects in endocytosis, cell polarization, bud site
selection in diploid cells, and a depolarized actin cytoskeleton (7, 8,
10). Rvs161p and Rvs167p have been shown to interact through the BAR
domain in the two-hybrid system (11). However, their localization in
the cell seems to be different, raising the question whether the
Rvs161p-Rvs167p interaction is relevant in vivo. Rvs161p was
shown to be mainly cytosolic in unbudded cells, and upon bud emergence
it localizes mainly to the mother-bud neck region (9). In contrast, in
unbudded cells Rvs167p is localized mainly in small cortical patches
throughout the cell which polarize at the bud emergence site and in
small buds (12). By using the two-hybrid system the BAR domain of Rvs167 has also been shown to mediate homodimerization in one study
(13), and another study failed to detect any Rvs167p-Rvs167p interaction (11).
Mutations in rvs161 and rvs167 affect the actin
cytoskeleton (7, 10). Interestingly, the SH3 domain of Rvs167p has been shown to interact with actin in the two-hybrid system (14). The finding
that Rvs167p interacts with the actin-binding protein Abp1p through its
GPA/SH3 domains (15) led to the hypothesis that Abp1p could mediate the
interaction between Rvs167p and actin.
In this study we investigated whether Rvs161p and Rvs167p indeed
function together in vivo. We find that the steady state level of Rvs161p is strongly reduced in an rvs167
strain.
Similarly, the level of Rvs167p is strongly reduced in an
rvs161
strain. We demonstrate that these reduced protein
levels at steady state are caused by a decreased stability of either
Rvs protein in a strain mutated in the other rvs gene.
Furthermore, we provide evidence that the amount and ratio of Rvs161p
and Rvs167p are critical parameters for receptor-mediated endocytosis.
In addition, by using the two-hybrid system we find that Abp1p is not
required to mediate the interaction of Rvs167p with actin.
 |
MATERIALS AND METHODS |
Yeast Strains, Media, and General Techniques--
Yeast strains
used in this study were EGY48 (Mat a his3 trp1 ura3
leu2::lexAop6-LEU2), RH448 (Mat a his4 leu2 lys2
ura3 bar1), RH3376 (Mat a his3 leu2 trp1 ura3 bar1),
RH2600 (Mat a his4 ura3 rvs161
bar1), RH2950
(Mat a his4 leu2 trp1::URA3 ura3 rvs167::TRP1 bar1), RH5238 (Mat a his3 trp1 ura3
leu2::lexAop6-LEU2 abp1::KanMX6), and RH5239
(Mat a his3 trp1 ura3 leu2::lexAop6-LEU2
rvs161::KanMX6). Strains that did not bear plasmids were
grown in complete media YPUAD (2% glucose, 2% peptone, 1% yeast
extract, 40 µg/ml uracil, 40 µg/ml adenine, and 40 µg/ml
tryptophan, 2% agar for solid media). Unless mentioned otherwise,
strains bearing plasmids were selected on SD minimal media (16).
Standard recombinant DNA techniques were used (17). Restriction
endonucleases were obtained from MBI Fermentas; Klenow was from Roche
Molecular Biochemicals, and Pfu polymerase was obtained from Stratagene.
Plasmid Constructions--
pEG202 and pJG4-5 were described
elsewhere (18); both plasmids contain a 2-µ origin of
replication. The reporter gene plasmid pSH18-34 was described elsewhere
(19), and it contains eight LexA operators upstream of the reporter
gene GAL1-lacZ. To construct pEGRVS161
the RVS161-ORF1
was amplified by the polymerase chain reaction with
Pfu polymerase using the 5'-primer
GATACGGAATTCATGAGTTGGGAAGGTTTTAAG and 3'-primer GAGTATTCCGCTCGAGTTATTTTATCCCGAGCGCACAAAT introducing an
EcoRI site upstream and an XhoI site downstream
of the RVS161-ORF. The fragment was then inserted as an
EcoRI-XhoI fragment into pEG202. To construct
pJGACT1 the ACT1-ORF was amplified by the
polymerase chain reaction using the 5'-primer
GATACGGAATTCATGGATTCTGGTATGTTCTAG and 3'-primer
TACGCGGATCCTTAGAAACACTTGTGGTGAACG introducing an EcoRI site
upstream and a BamHI site downstream of the
ACT1-ORF. The fragment was then cut with BamHI,
the 5' overhang filled in with Klenow, and cut with EcoRI.
This fragment was then inserted in a pJG4-5 which was cut with
XhoI, the 5' overhang filled in with Klenow, and cut with
EcoRI. To construct pEGRVS167 the
RVS167-ORF was amplified by the polymerase chain reaction
using the 5'-primer RVS167,1 (GATACGGAATTCATGAGTTTTAAAGGGTTTACCAAG) and
3'-primer RVS167,2 (GAGTATTCCGCTCGAGCTAGTTCTTGTTGAGTTGCACG) introducing an EcoRI site upstream and an XhoI site
downstream of the RVS167-ORF. The fragment was then inserted
as an EcoRI-XhoI fragment into pEG202.
pJGRVS167 was described elsewhere (20). To construct pJGBAR,
the BAR domain of RVS167 was amplified by the polymerase chain reaction using the 5'-primer RVS167,1 and 3'-primer
GAGTATTCCGCTCGAGCTACTTAGAGTAACCGAGTTTAAAG introducing an
EcoRI site upstream and an XhoI site downstream of the BAR domain. The fragment was then inserted as an
EcoRI-XhoI fragment into pJG4-5. To construct
pJGBAR/GPA the BAR and GPA domains of RVS167 were amplified
by the polymerase chain reaction using the 5'-primer RVS167,1 and
3'-primer RVS167,6 (GAGTATTCCGCTCGAGCTAGCCAGGAGCTGCCGCTACG) introducing
an EcoRI site upstream and an XhoI site
downstream of the BAR/GPA domains. The fragment was then inserted as an
EcoRI-XhoI fragment into pJG4-5. To construct
pJGGPA, the GPA domain of RVS167 was amplified by the
polymerase chain reaction using the 5'-primer RVS167,8
(GATACGGAATTCCTTTAAACTCGGTTACTCTAAG) and 3'-primer RVS167,6 introducing
an EcoRI site upstream and an XhoI site
downstream of the GPA domain. The fragment was then inserted as an
EcoRI-XhoI fragment into pJG4-5. To construct
pJGGPA/SH3 the GPA and SH3 domains of RVS167 were amplified
by the polymerase chain reaction using the 5'-primer RVS167,8 and
3'-primer RVS167,2 introducing an EcoRI site upstream and an
XhoI site downstream of the GPA/SH3 domains. The fragment
was then inserted as an EcoRI-XhoI fragment into
pJG4-5. To construct pJGSH3, the SH3 domain of RVS167 was amplified by the polymerase chain reaction using the 5'-primer GATACGGAATTCCCTGGCGTTGAAACTGTTACCG and 3'-primer RVS167,2
introducing an EcoRI site upstream and an XhoI
site downstream of the SH3 domain. The fragment was then inserted as an
EcoRI-XhoI fragment into pJG4-5. To construct
p181RVS161 and p195RVS161, the 2.2-kilobase pair
EcoRI fragment from pUB1-3 (8) was excised and cloned into
Yeplac181 (p181 (21)) and Yeplac195 (p195 (21)). To construct p181RVS167 and p195RVS167, the 1.8-kilobase pair
KpnI-SphI fragment from pFBKS (7) was excised and
cloned into Yeplac181 (p181) and Yeplac195 (p195).
Two-hybrid Analysis--
Two-hybrid analysis was performed as
described (22). Briefly, the strains contain the reporter plasmid
pSH18-34 with the reporter gene GAL1-lacZ under
the control of eight LexA operators. To assay the interaction between
bait and prey, the strains are streaked out on plates containing X-gal.
The positive colonies turn dark, and the negative ones remain white.
Protein Extracts and Immunoblotting--
Yeast strains were
grown to exponential phase, harvested, and lysed with glass beads in
500 µl of lysis buffer (50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 0.5% SDS, and 1 µg/ml of the protease
inhibitors aprotinin, leupeptin, pepstatin A, chymostatin, and
antipain). The protein concentration of the lysates was measured using
the Bio-Rad protein assay. Equivalent amounts of each sample were analyzed by SDS-PAGE (23) and immunoblotting with antibodies against
Rvs161p and Rvs167p. As a control, antibodies against Sla2p were used
(24).
Pulse-Chase Experiments--
Metabolic labeling of cells was
carried out as described previously (8) with the modification that the
cells were not converted to spheroplasts but were lysed with glass
beads in 200 µl of lysis buffer and then heated for 3 min at
95 °C. Rvs161p or Rvs167p were immunoprecipitated from the lysates
and analyzed by SDS-PAGE and autoradiography.
Coimmunoprecipitation--
WT (RH448) and rvs167
(RH2950)
strains were grown to exponential phase, harvested, and converted to
spheroplasts using lyticase (25). The spheroplasts were lysed by
osmotic shock in 2 ml of buffer (20 mM MES pH 6.5, 100 mM NaCl, 5 mM MgCl2, 1% Nonidet P-40, 0.5 mM phenylmethylsulfonyl fluoride, and 1 µg/ml
of the protease inhibitors pepstatin, leupeptin, antipain). The protein concentration of the lysates was measured using the Bio-Rad protein assay. Equal amounts of total protein for both strains were taken, and
proteins were immunoprecipitated with antibodies against Rvs167p. The
immunoprecipitates were analyzed by SDS-PAGE and immunoblotting with
antibodies against Rvs161p.
-Factor Uptakes--
-Factor uptakes (continuous presence)
were done as described previously (16). The strains were tested at
24 °C. The internalization rates were calculated as the percentage
of counts internalized per time unit in the linear range and normalized
to the internalization rate of the strain with the empty vector set to
100%. The results represent the average of at least three independent
experiments, and the S.D. was calculated.
Growth Curves--
The strains transformed with the indicated
plasmids were grown overnight, and the cell density was determined. The
cultures were diluted to about 106 cells/ml; a sample was
taken (time point 0), and the cultures were incubated at 24 °C.
Every 3 h a sample was taken and the cell density determined.
Actin Staining--
Yeast cell pre-cultures were grown at
24 °C in SD selective media to maintain the plasmids. Cells taken
from the pre-culture were then grown at 24 °C in YPUAD to early log
phase. Cells at 1.5 × 107 were then fixed in
formaldehyde and stained with TRITC-phalloidin (Sigma) to visualize
F-actin essentially as described previously (26).
 |
RESULTS |
Interactions of the BAR Domain of Rvs167p--
Rvs167p can be
divided into three regions as follows: the N-terminal BAR domain, the
GPA domain, and the C-terminal SH3 domain (Fig.
1). Two previous studies have shown that
the BAR domain of Rvs167p interacts with Rvs161p. However, in one of
these reports the BAR domain of Rvs167p also interacted with
full-length Rvs167p (13), and in the other report no such interaction
was detected (11). To clarify this point we tested these interactions
in a different two-hybrid system. Both previous studies used a
two-hybrid system with the yeast Gal4p as DNA-binding domain, whereas
in our system the Escherichia coli protein LexA is used as
DNA-binding domain. As expected we detected a strong interaction of
Rvs161p with the BAR domain of Rvs167p (Fig.
2A). As shown in Fig.
2B we also detected a strong interaction between full-length
Rvs167p and the BAR domain of Rvs167p. Since the BAR domain of Rvs167p mediates both the interactions with Rvs161p and Rvs167p, there is the
possibility that the interaction of Rvs167p with itself is indirect and
mediated via Rvs161p. To test this we performed a two-hybrid analysis
in an rvs161
strain, and we still detected a strong
Rvs167p-Rvs167p interaction (Fig. 2C), suggesting that the
interaction is direct or mediated by a protein other than Rvs161p. To
confirm the report that Rvs161p and Rvs167p form a complex in
vivo (11), we showed that the two proteins could be
coimmunoprecipitated under native conditions with antibodies against
Rvs167p (Fig. 2D).

View larger version (5K):
[in this window]
[in a new window]
|
Fig. 1.
Schematic overview of the domains of Rvs161p
and Rvs167p. BAR,
BIN/Amphiphysin/RVS domain;
GPA, glycine-proline-alanine-rich region; SH3,
Src homology 3 domain.
|
|

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 2.
Two-hybrid interactions of the BAR domain of
Rvs167p. Strains containing the reporter gene plasmid pSH18-34
with the lacZ gene under the control of eight LexA
operators, a bait, and a prey were streaked out on plates containing
X-gal. Positive interactors turn dark and negative colonies remain
white. A, full-length Rvs161p as bait (pEGRVS161)
was tested against full-length Rvs167p (pJGRVS167) and
indicated Rvs167p domains as preys. B, full-length Rvs167p
as bait (pEGRVS167) was tested against full-length Rvs167p
(pJGRVS167) and indicated Rvs167p domains as preys.
C, the interaction of Rvs167p with itself was tested in a WT
(EGY48) and in an rvs161 (RH5239) strain. D,
coimmunoprecipitation of Rvs161p and Rvs167p. Lysates from WT (RH448)
and rvs167 (RH2950) were incubated with antibodies
against Rvs167p or with a preimmune serum, and the immunoprecipitates
were analyzed by SDS-PAGE and immunoblotting with antibodies against
Rvs161p.
|
|
Reduced Stability of Rvs Proteins in the Absence of Its
Partner--
Interestingly, the localization of Rvs161p (mostly
cytoplasmic, at mother-bud neck region in small budded cells) and
Rvs167p (small cortical patches that polarize upon bud emergence) seems to be different (9, 12). These findings raise the question whether the
interaction of Rvs161p with Rvs167p is required in vivo. We
found that when compared with a WT strain at steady state, the level of
Rvs167p is strongly reduced in an rvs161
strain (Fig.
3A). Similarly, the level of
Rvs161p is strongly reduced in an rvs167
strain (Fig.
3A). As control we detected Sla2p, a protein that is also
required for endocytosis and actin organization (24). Its levels were
constant in the three strains (Fig. 3A). A decreased
synthesis or an increased instability of the proteins in the mutant
strains could cause these reduced protein levels at steady state. To
address this question we performed pulse-chase experiments in the
different strains. As shown in Fig. 3B, both Rvs161p in an
rvs167
strain and Rvs167p in an rvs161
strain are unstable when compared with a WT strain. Taken together
these data suggest an in vivo function for the
Rvs161p-Rvs167p interaction in stabilizing both proteins.

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 3.
Interaction of Rvs161p and Rvs167p is
required for the stability of both proteins. A, protein
extracts from WT (RH3376), rvs161 (RH2600), and
rvs167 (RH2950) at steady state were analyzed by SDS-PAGE
and immunoblotting with antibodies against Rvs161p and Rvs167p. As a
control the same extracts were blotted with antibodies against Sla2p.
B, pulse-chase experiments. WT (RH3376),
rvs161 (RH2600), and rvs167 (RH2950) cells
were metabolically labeled, and lysates were prepared as described
under "Materials and Methods." Proteins were immunoprecipitated
from the lysates with antibodies against Rvs161p or Rvs167p, and the
immunoprecipitates were analyzed by SDS-PAGE and autoradiography.
|
|
Amount and Ratio of Rvs161p and Rvs167p Are Critical Parameters for
Endocytosis--
Mutations in rvs161 or rvs167
have both been shown to affect the internalization step of
receptor-mediated endocytosis (8). To learn more about the involvement
of the proteins in endocytosis, we decided to overexpress them and
measure how they affect
-factor internalization. Introduction of a
2-µ plasmid with RVS161 or RVS167 in a WT
strain does not affect
-factor internalization (Fig.
4A). Interestingly,
overexpression of Rvs167p in an rvs161
strain exacerbated
the endocytic defect of this strain (Fig. 4B). Similarly,
overexpression of Rvs161p in an rvs167
strain exacerbated the endocytic defect of this strain (Fig. 4C). The protein
levels of Rvs161p and Rvs167p upon overexpression in a WT or a mutated strain are similar (data not shown). Interestingly, co-overexpression of both Rvs161p and Rvs167p in a WT strain reduces
-factor
internalization to about two-thirds of WT rate (Fig. 4D).
Taken together these data suggest that not only the amounts of Rvs161p
and Rvs167p but also their ratio are critical parameters for
endocytosis. We have tested the same strains overexpressing the Rvs
proteins for an actin and a growth phenotype. As shown in Fig.
5, A-H, overexpression of the
Rvs proteins either individually or together in a WT strain does not
affect the actin cytoskeleton profoundly. The cells have a normal
polarized actin cytoskeleton with cables running along the mother-bud
axis. Also the growth rates of the different strains are not greatly
affected (Fig. 5I). As shown in Fig.
6, A-H, overexpression of
Rvs167p in an rvs161
strain or overexpression of Rvs161p
in an rvs167
strain does not further deteriorate or
ameliorate the actin defect exhibited by the mutants. We detect a mild
growth phenotype upon overexpression of Rvs167p in the
rvs161
strain when compared with the strain with the
empty vector (Fig. 6I). Overexpression of Rvs161p in an
rvs167
strain has no obvious effect on the growth rate
(Fig. 6I).

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 4.
-Factor uptakes. A,
WT strain (RH3376) transformed with either p181, p181RVS161,
or p181RVS167 were tested for -factor internalization at
24 °C. The internalization rates were calculated as the percentage
of counts internalized per time unit in the linear range and normalized
to the strain with p181 set to 100%. Note that the internalization
rate of the WT strain transformed with p181 is ~4.6%/min.
B, rvs161 strain (RH2600) transformed with
either p195 or p195RVS167 were tested for -factor
internalization at 24 °C. The internalization rates were calculated
as the percentage of counts internalized per time unit in the linear
range and normalized to the strain with p195 set to 100%. Note that
the internalization rate of the rvs161 strain transformed
with p195 is ~1.7%/min. C, rvs167 strain
(RH2950) transformed with either p181 or p181RVS161 were
tested for -factor internalization at 24 °C. The internalization
rates were calculated as the percentage of counts internalized per time
unit in the linear range and normalized to the strain with p181 set to
100%. Note that the internalization rate of the rvs167
strain transformed with p181 is ~2.0%/min. D, WT strain
(RH3376) transformed with combinations of either p181, p195,
p181RVS167, and p195RVS161 were tested for
-factor internalization at 24 °C. The internalization rates were
calculated as the percentage of counts internalized per time unit in
the linear range and normalized to the strain with p181/p195 set to
100%.
|
|

View larger version (55K):
[in this window]
[in a new window]
|
Fig. 5.
Actin staining and growth curve.
A-H, WT strain (RH3376) transformed with p181 and p195
(A and B), p181 and p195RVS161
(C and D), p181RVS167 and p195
(E and F), p181RVS167 and
p195RVS161 (G and H) were fixed, and
filamentous actin was visualized using TRITC-phalloidin (A, C,
E, and G) or Nomarski optics (B, D, F, and
H). I, the same strains were grown overnight. The
cultures were diluted to about 106 cells/ml and incubated
at 24 °C, and the cell densities were determined every 3 h.
|
|

View larger version (60K):
[in this window]
[in a new window]
|
Fig. 6.
Actin staining and growth curve.
A-D, rvs161 strain (RH2600) transformed with
either p195 (A and B) or p195RVS167
(C and D) were fixed, and filamentous actin was
visualized using TRITC-phalloidin (A and C) or
Nomarski optics (B and D). E-H,
rvs167 strain (RH2950) transformed with either p181
(E and F) or p181RVS161 (G
and H) were fixed, and filamentous actin was visualized
using TRITC-phalloidin (E and G) or Nomarski
optics (F and H). I, the same strains
were grown overnight. The cultures were diluted to about
106 cells/ml and incubated at 24 °C, and the cell
densities were determined every 3 h.
|
|
Interaction of Rvs167p with Actin Does Not Require
Abp1p--
Mutations in rvs167 affect the actin
cytoskeleton (7), and Rvs167p has been shown to interact with actin via
its SH3 domain (14). The actin-binding protein Abp1p has been proposed
to mediate this interaction of Rvs167p and actin since it interacts
with the GPA/SH3 domains of Rvs167p (15). As shown in Fig.
7, the interaction of Rvs167p with actin
in the two-hybrid system is not abolished in an abp1
strain showing that Abp1p is not required to mediate the interaction of
Rvs167p with actin. Interestingly, we also detect an interaction of
Rvs161p with actin in the two-hybrid system. However, this interaction
is abolished in an rvs167
strain suggesting that the
interaction of Rvs161p with actin is mediated via Rvs167p (data not
shown).

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 7.
Two-hybrid analysis. The interaction of
Rvs167p (pEGRVS167) with actin (pJGACT1) was
tested in a WT (EGY48) and in an abp1 (RH5238) strain.
The strains contain the reporter gene plasmid pSH18-34 with the
lacZ gene under the control of eight LexA operators, a bait
and a prey. They were streaked out on plates containing X-gal. Positive
interactors turn dark whereas negative colonies remain white.
|
|
 |
DISCUSSION |
In this study we provide direct evidence that Rvs161p and Rvs167p
function together in vivo. We find that the steady state levels of the two proteins are interdependent. This effect is caused by
a dramatically decreased stability of either Rvs protein in the absence
of its partner. These data provide evidence that the interaction of
Rvs161p and Rvs167p is physiologically relevant and required for the
stability of both proteins. Furthermore, these findings might explain
the almost identical phenotypes that are seen upon mutation of the two
genes individually. The mutant phenotypes detected in either mutant
strain might be a combination of loss of both Rvs proteins.
Nevertheless, the function of Rvs161p in cell fusion (9) does
not seem to require Rvs protein-protein interaction. Apparently, the
highly reduced levels of Rvs161p in the rvs167
mutant are
sufficient for this function. As seen in Fig. 3B, Rvs161p is
more sensitive to Rvs167p levels than vice versa. Since Rvs167p has two
additional domains when compared with Rvs161p, this difference might
reflect a partial stabilization of Rvs167p in the absence of Rvs161p
via interactions mediated by these domains with other proteins
(e.g. interaction with actin).
As mentioned in the Introduction, two previous studies have
investigated the cellular localization of Rvs161p (mostly cytoplasmic, at mother-bud neck region in small budded cells (9)) and Rvs167p (small
cortical patches that polarize upon bud emergence (12)). The two
proteins apparently do not colocalize in the cell; however, in this
study we have provided evidence that these two proteins do function
together in vivo. This discrepancy might be explained if one
considers that Rvs161p localization is mainly cytosolic. Therefore, it
might also be localized to the Rvs167p-containing patches but is not
concentrated there. In the case of Rvs167p, the bright fluorescence
caused by its high concentration in cortical patches might make it
difficult to detect an additional weak diffuse cytosolic staining.
Therefore, it is possible that a certain amount of Rvs167p is localized
to the cytosol and interacts with Rvs161p.
Interestingly, overexpression of either Rvs161p or Rvs167p alone in a
WT strain has no major effect on the internalization step of
endocytosis. However, co-overexpression of both proteins reduced the
-factor internalization rate to two-thirds of WT levels. Also,
overexpression of Rvs161p in an rvs167
strain, as well as
overexpression of Rvs167p in an rvs161
strain,
exacerbated the endocytic defect found in the mutated strains. Since
the amount of protein upon overexpression is similar in all the strains
(WT, rvs161
, and rvs167
, data
not shown), we conclude that both the amount and the ratio of Rvs161p
and Rvs167p are critical parameters for the internalization step of
endocytosis. We have tested the strains used for the endocytosis assays
for an actin and a growth phenotype. None of the strains overexpressing
the Rvs proteins exhibited an obvious change in their actin
cytoskeleton when compared with the strains with the empty vectors.
Also the growth rates were almost identical with the exception
of a mild defect detected upon overexpression of Rvs167p in
the rvs161
strain. The endocytic defect we
detect in some of the strains overexpressing the Rvs proteins
might be explained in two ways. One possibility would be that
overexpression of the Rvs proteins causes a very subtle actin
defect not detectable by immunofluorescence but affecting endocytosis.
Another possibility would be that by overexpressing the Rvs proteins
another protein required for endocytosis is titrated out and therefore
endocytosis is affected.
By using the two-hybrid system, we detected an interaction of Rvs167p
with itself mediated via the BAR domain as previously described (13).
Since the same domain also mediates the interaction with Rvs161p, we
wanted to determine whether the Rvs167p-Rvs167p interaction is direct
or mediated via Rvs161p. Two-hybrid analysis in an rvs161
strain demonstrated that Rvs161p is not required for this interaction
and, therefore, Rvs167p either interacts directly with itself or the
interaction is mediated via a protein other than Rvs161p.
Interestingly, amphiphysin I has also been shown to interact with
itself in mammalian cells (27).
Mutations in rvs161 and rvs167 have been shown to
affect the actin cytoskeleton (7, 10). A previous study has shown that Rvs167p interacts with actin via its SH3 domain (14), and it was
suggested that the actin-binding protein Abp1p could mediate this
Rvs167p-actin interaction (15). Here we show that in the two-hybrid
system Rvs167p interacts with actin in an abp1
strain showing that Abp1p is not required for this interaction. There are
several possible explanations for this finding. First, it could be that
in the absence of Abp1p another protein takes over its function.
Second, there could be more than one protein involved in mediating this
interaction. Third, Rvs167p could interact directly with actin. Another
potential candidate to mediate the Rvs167p-actin interaction could be
Las17p, since it was shown to interact with the GPA/SH3-domains of
Rvs167p in the two-hybrid system (32).
rvs mutant cells show pleiotropic phenotypes including
sensitivity to nonoptimal growth conditions, defects in the
organization of the actin cytoskeleton, defects in bud-site selection,
and endocytosis (7, 8, 10). Interestingly, Rvs167p was identified as a
target of the Pho85 cyclin-dependent kinase and thus
might be involved in the regulation of the actin cytoskeleton early in
cell cycle (28). Also its mammalian homolog, amphiphysin, has been
shown to be regulated by phosphorylation (27, 29). In addition,
amphiphysin I has been implicated in the autoimmune Stiff-Man syndrome
disorder associated with breast cancer, and amphiphysin II has been
shown to interact with the MYC oncoprotein implicating it in cell cycle
control (2, 30). Taken together these data suggest that some general
functions and regulation of these proteins have been conserved through
evolution. In addition, these findings implicate the members of this
protein family not only as important players in intracellular membrane
trafficking but also in mediating signals during the cell cycle.
In summary, we demonstrate for the first time that the yeast
amphiphysin homologs Rvs161p and Rvs167p function together in vivo. The
interaction between the proteins seems to be crucial for their
stability. Furthermore, we show that the amount and ratio of Rvs161p
and Rvs167p are critical parameters for the internalization step of endocytosis.