From the Institute of Molecular Biology, University of Oregon,
Eugene, Oregon 97403-1229
Membrane traffic in eukaryotic cells requires the
interaction of a vesicle-associated soluble NSF attachment protein
receptor (v-SNARE) on transport vesicles with a SNARE on the target
membrane (t-SNARE). Recently, we identified the yeast protein Vti1p as a v-SNARE that is involved in two transport reactions. Vti1p interacts with the prevacuolar t-SNARE Pep12p in Golgi to prevacuolar transport and with the cis-Golgi t-SNARE Sed5p in traffic to the cis-Golgi. Here
we describe a human Vti1p homolog, hVti1. Whereas vti1
cells are inviable, expression of hVti1 allows vti1
cells to grow at nearly the wild-type growth rate. When expressed in
yeast hVti1 can replace Vti1p in both Golgi to prevacuolar transport
and in traffic to the cis-Golgi. Sequence comparisons with a
Schizosaccharomyces pombe and two different mouse Vti1
homologs led to the identification of a very conserved predicted
-helix. Amino acid exchanges in vti1 mutant alleles
defective either in one or both trafficking steps cluster in this
domain, suggesting that this structure is probably the binding site for
effector proteins.
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INTRODUCTION |
Transport between many different organelles in eukaryotic cells
occurs via transport vesicles, which must have the ability to recognize
their target membranes.
SNARE1 proteins provide this
information (1, 2). In the SNARE model, a specific set of v-SNAREs
localized on transport vesicles interacts with specific t-SNAREs on the
target membrane. Both v- and t-SNAREs contain a single C-terminal
transmembrane domain and predicted coiled coil domains. It has been
demonstrated that t-SNAREs interact via their coiled coil domains with
v-SNAREs. A growing number of SNARE proteins have been identified from
both yeast and mammalian cells. SNARE proteins involved in identical membrane trafficking steps in yeast and mammals share significant amino
acid identities. In yeast the v-SNAREs Sec22p (Sly2p), Bet1p (Sly12p),
Bos1p, and Ykt6p are involved in transport from the ER to the cis-Golgi
compartment (3-5). Their t-SNARE partner in the cis-Golgi compartment
is Sed5p (6). It has been demonstrated that in addition to interactions
with the anterograde v-SNAREs, Sed5p also binds to the medial Golgi
v-SNARE Sft1p, which is involved in retrograde traffic from the medial
to the cis Golgi compartment (7). Recently, Sec22p has been found in a
complex with the ER t-SNARE Ufe1p and has been implicated in retrograde
traffic to the ER (8). Mammalian homologs have been identified for Sec22p, rsec22 and ERS-24, and for Bet1p, rbet1 (9, 10). ERS-24
interacts with syntaxin 5, the mammalian homolog of Sed5p (11).
Proteins traversing the secretory pathway are sorted in the trans-Golgi
network according to their destination (12). In mammalian cells,
vesicles of the constitutive and regulated secretory pathway destined
for the plasma membrane bud from the trans-Golgi network. Soluble
lysosomal proteins are marked by a mannose 6-phosphate residue and bind
to the mannose 6-phosphate receptor (13). The complex leaves in
transport vesicles targeted to the late endosomal compartment, from
which the mannose 6-phosphate receptor is recycled. In yeast about 50 VPS and PEP genes have been identified, which function in traffic from the late Golgi compartment to the vacuole, the
mammalian equivalents of the trans-Golgi network and the lysosome respectively (14-17). The soluble vacuolar hydrolase carboxypeptidase Y (CPY) binds to the CPY-receptor Vps10p in the late Golgi compartment via a peptide sorting signal (18). The complex is transported to the
prevacuolar compartment where it dissociates. CPY is transported on to
the vacuole and Vps10p is recycled (19-21). Pep12p has been identified
as a t-SNARE residing in the prevacuolar compartment (22). The
mammalian protein syntaxin 6 displays 25% amino acid identity with
Pep12p, is localized in the Golgi region, and has been proposed to be
the functional homolog of Pep12p (23). A Pep12p homolog with 32% amino
acid identity has been identified in Arabidopsis, which can
restore at least some vacuolar protease function to pep12
mutants in yeast (24).
Recently, we described a yeast v-SNARE, Vti1p, that is essential for
yeast cell viability (25). The temperature-sensitive vti1
mutants vti1-1 and vti1-2 are defective in late
Golgi to prevacuolar transport of CPY. Genetic interactions between
VTI1 and PEP12 as well as physical association of
Vti1p and Pep12p indicate that Vti1p and Pep12p form a v-SNARE-t-SNARE
complex. A second class of temperature-sensitive mutants, such as
vti1-11, display a severe growth defect and a block in
traffic to the cis-Golgi compartment, in addition to a block in late
Golgi to prevacuolar transport of CPY. Overexpression of Sed5p
suppressed the cis-Golgi traffic block but had no effect on the defect
in sorting proteins to the vacuole. Recombinant Vti1p and Sed5p
interacted in vitro. We have proposed that Vti1p forms a
SNARE complex with Sed5p in retrograde traffic either from the
prevacuolar compartment or from the late Golgi compartment.
Here we describe the identification of a human Vti1 homolog. hVti1 is
able to function in both traffic steps that require Vti1p in yeast.
Conserved amino acid residues between the yeast and human proteins
together with mutations in yeast VTI1 have led us to propose
a structural model, in which a predicted
-helical coiled coil domain
in Vti1p interacts with both Pep12p and Sed5p.
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EXPERIMENTAL PROCEDURES |
Materials--
Reagents were used from the following sources:
enzymes for DNA manipulation from New England Biolabs (Beverly, MA) and
Boehringer Mannheim, [35S]Express label from NEN Life
Science Products, fixed Staphylococcus aureus cells
(IgGsorb) from The Enzyme Center (Malden, MA), and Oxalyticase from
Enzogenetics (Corvallis, OR). The human hypothalamus cDNA library
in
ZAPII was obtained from the ATCC (Rockville, MD). All other
reagents were purchased from Sigma. Yeast strains (Table
I) were grown in rich medium (1% yeast
extract, 1% peptone, 2% dextrose, YEPD) or standard minimal medium
(SD) with appropriate supplements. To induce expression from the
GAL1 promoter, dextrose was replaced by 2% raffinose and
2% galactose. Plasmid manipulations were performed in the
Escherichia coli strains MC1061 or XL1Blue using standard
media (Table II).
Plasmids and Strains--
hVti1 was PCR-amplified from a human
glioblastoma cDNA library in pADANS (26) using an oligonucleotide
complimentary to the ADH terminator (5
-AAC CTC TGG CGA AGA AGT CCA-3
)
and complimentary to sequences 3
of the hVti1-coding region (5
-CAG
CCC ACA GCA ATA TGC-3
). The resulting 900-base pair fragment was
gel-purified and cloned into EcoRV digested pBluescript
KS+ to obtain pFvM46. To isolate pFvM60 and pFvM61, hVti1
was PCR-amplified from a human hypothalamus cDNA inserted into
ZAPII (27) using a T7 oligonucleotide and the same 3
hVti1
oligonucleotide, the fragment was gel purified, digested with
SacI and XhoI, and cloned into pBluescript
KS+. hVti1 (codons 62-232) was PCR-amplified from pFvM46
with the oligonucleotides 5
-CCG CTC GAG ATG GAG GAG GAG CTA C-3
and
5
-CGG GAT CCT ACG CAT AGT CAG GAA CAT CAT ATG GGT AAT GGC TGC GAA AGA ATT TG-3
and cloned into the multicopy yeast expression vector pVT100-U behind the ADH1 promoter (28) to obtain pFvM58. To construct pFvM118, hVti1 (codons 1-232) was PCR-amplified from pFvM50
using the oligonucleotide 5
-CCG CTC GAG ATG GCC TCC TCC GCC GCC TC-3
and the same 3
oligonucleotide as for pFvM58, and cloned into
pVT100-U. pFvM91 encoding vti1-12 was generated by random
PCR mutagenesis and was isolated in a screen for temperature sensitive
growth defects in the same way as vti1-11 (25). The 2.8-kb
ScaI-BglII fragment from pFvM28 (encoding wild
type VTI1) was ligated with the 3.9-kb
ScaI-BglII fragment of pFvM92 to construct pFvM108 (wild type N terminus codon 1-105, vti1-11 mutant C
terminus codon 106-217). pFvM116 was constructed by subcloning a
2.9-kb EcoRI-SacI fragment from pRB58 (29)
encoding SUC2 into pRS315 (30). vti1-11 from
pFvM108 was subcloned into the integration vector pRS306 (30). FvMY21
was constructed by integration of this plasmid linearized by
XbaI into SEY6211 and looping out the wild type
VTI1 on 5-fluorourotic acid plates (31).
Screen for Human vti1
Suppressors--
To identify human
proteins that allow for growth in the absence of Vti1p, the strain
FvMY6/pFvM16 (vti1
/pCEN-GAL1-VTI1) was used.
FvMY6/pFvM16 cells were transformed with yeast expression plasmids
encoding human proteins. This library consisted of human glioblastoma
cDNAs fused to the ADH promoter and the first 14 ADH1 codons in the multicopy yeast vector pADANS (26).
Transformants were plated on SD-Leu plates, conditions that turn off
the expression of VTI1 and prevent growth of FvMY6/pFvM16
cells because Vti1p is essential for growth. Colonies that grew were
tested for the absence of Vti1p by immunoblot analysis. Plasmids were
recovered and retransformed, and colonies that lost the pFvM16 plasmid
were selected to confirm that suppression was dependent on the
expression of a human protein. The insert of the suppressor plasmid
pFvM50 was sequenced and encoded a human Vti1p homolog.
Immunoprecipitation of 35S-Labeled Proteins--
The
procedures for CPY and invertase immunoprecipitation were described
earlier (25, 32, 33). For CPY immunoprecipitations yeast cells were
grown at 22 or 30 °C, radiolabeled with
[35S]methionine for 10 min at the indicated temperature,
and chased for 30 min after addition of 500 µg/ml methionine and
cysteine. Invertase was derepressed by an incubation in minimal medium
containing 0.1% glucose, 50 mM KPO4, pH 5.7, and 1 mg/ml bovine serum albumin for 30 min at 22 °C plus 15 min at
37 °C. Cells were radiolabeled for 7 min at 37 °C and chased for
0 min or 30 min. Cells were spheroplasted, and extracts were prepared
from internal and external fractions, and CPY- or
invertase-immunoprecipitated with polyclonal antibodies and fixed
S. aureus cells. Immunoprecipitates were analyzed by
SDS-polyacrylamide gel electrophoresis and autoradiography.
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RESULTS |
Identification of a Human Vti1p Homolog--
A multicopy
suppressor screen was used to identify human proteins that allow for
survival of yeast cells in the absence of Vti1p. vti1
cells expressing VTI1 under the control of the
GAL1 promoter were transformed with the multicopy library
pADANS. The pADANS library contains human glioblastoma cDNAs fused
to the ADH1 promoter and the first 14 ADH1
codons, which results in expression of human fusion proteins in yeast
(26). In the resulting transformants, expression of VTI1 was
turned off by plating the cells on media containing glucose. Colonies
that grew were tested for the absence of Vti1p by immunoblot analysis.
Plasmids were recovered from these suppressor strains, and
retransformed into vti1
GAL-VTI1 cells. Colonies that
lost the GAL1-VTI1 plasmid were selected to confirm that
suppression was dependent on the expression of a human protein.
Sequencing of the suppressor (pFvM50) revealed that the clone is
predicted to encode a 232-amino acid protein with a C-terminal
transmembrane domain. This protein displays 29% overall amino acid
identity with the yeast Vti1p (25), and was therefore called hVti1
(Fig. 1A). The 56-amino acid
domain adjacent to the transmembrane domain is more homologous (41%
amino acid identity). The regions between amino acid 38 and 67 and
amino acid 160 and 193 are predicted to form amphipathic
-helical
coiled coils using the paircoil program (probability score 0.71 and
0.69) (34). The region in yeast Vti1p homologous to the second
predicted coiled coil region is also predicted to form a coiled coil
domain, indicating that the proteins may adopt similar structures. The first structural studies with SNARE proteins indicate that the isolated
t-SNAREs SNAP-25 and Sec9p and the v-SNARE Snc1p are largely
unstructured (35, 36). A large increase in
-helical contents was
observed after formation of SNARE complexes. Complex formation may
induce similar structural changes in Vti1p.

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Fig. 1.
Sequence comparison between different
Vti1 proteins. A, different Vti1 proteins were aligned using
the ClustalW program (50). The transmembrane domains are boxed,
predicted coiled coil regions marked with a solid line
(higher probability) or dashed line (lower probability)
above the sequence. Arrowhead, start of the short hVti1;
predicted fusion peptides between solid circles; conserved
predicted -helix between solid squares; amino acid
exchanges in yeast mutant Vti1 proteins: *, vti1-1; +,
vti1-2; , vti1-11; , vti1-12.
Accession nos.: HVti1, AF035824;
SpVti1, D89116; MVti1, AA240967,
AA016651, AA008789, AA105524, AA013839, AA086695, G1089688,
W53760, and W84286; MVti1b, AA097517,
AA016934, AA016379, and W13616. B, amino
acid identities and amino acid similarities taking conserved exchanges
into account. The GAP program was used for pairwise comparison.
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By searching a data base of human expressed sequence tags (EST), human
sequences that show high homology to the C-terminal half of the yeast
Vti1p were identified (accession nos. N39768 and R68750). These
sequences were identical to the C terminus of hVti1 identified in the
suppressor screen. An oligonucleotide primer complimentary to sequences
3
of the hVti1 and a primer containing vector sequences around the
polylinker were used to PCR amplify the hVti1 from two different human
cDNA libraries. One library consisted of human glioblastoma
cDNA in the yeast expression plasmid pADANS (26). The other was
human hypothalamus cDNA inserted into
ZAPII phage (27). DNA
sequences were amplified from both libraries, and these were predicted
to encode a 171 amino acid protein starting at methionine 62 of hVti1
(Fig. 1A, hVti1 starting at the arrowhead). A
clone derived from the
library (pFvM61) had an in-frame stop codon
upstream of the putative initiating methionine 62. A different
clone (pFvM60) and the clone derived from the pADANS library (pFvM 46)
contained a coding region for the same 171-amino acid long hVti1 but
were different upstream of the putative initiating methionine 62. pFvM46 ended after encoding 24 amino acids upstream of a putative
initiating methionine which are present in hVti1, indicating that it is
an incomplete clone. pFvM60 encoded 12 different amino acids upstream
of methionine 62 (AMSDFRSVCRRQ) and did not contain an upstream
in-frame stop codon.
Northern blot analysis of human RNA from different tissues revealed
that hVti1 was expressed as a single band of about 1.2 kb in all
tissues (data not shown). This suggests that hVti1 has a role in basic
cell function.
Further data base searches revealed the presence of a Vti1-related
hypothetical protein of unknown function in Schizosaccharomyces pombe, SpVti1 (Fig. 1A), which shares 38% amino acid
identity with S. cerevisiae Vti1p (Fig. 1B).
Recently, several Vti1p-related mouse ESTs were entered into the data
base. The ESTs were assembled into a mouse Vti1 protein (mVti1), which
is almost identical to hVti1 (93% amino acid identity). Surprisingly,
a second Vti1-related protein, mVti1b, could be assembled from a second
set of mouse ESTs. Unfortunately, the available sequences do not
include the stop codon and end directly before the expected
transmembrane domain. mVti1b shares only 31% amino acid identity with
mVti1 and about the same degree of amino acid identity with yeast Vti1p (35%). Three human ESTs with high identities to mVti1b were found in
the data base (AA326353, R29052, and T70362), indicating the presence
of a hVti1b, but the sequence data are not extensive enough to assemble
the full protein.
Structural Implication from the Vti1 Alignment and Analysis of vti1
Mutants--
In Vti1p the domain between amino acid 37 and 60 is
predicted to form an amphipathic
-helix which contains charged amino acids on one side and the other face is strongly hydrophobic due to the
presence of bulky hydrophobic amino acids (Fig.
2A). The homologous domains in
the other Vti1p-like proteins (Fig. 1, between filled
circles) also display similar properties. In hVti1 and mVti1 the
hydrophobic face is less pronounced but the domain between amino acid
residues 77 and 98 can also form an amphipathic
-helix. These
domains resemble amphipathic
-helical fusion peptides found in viral
fusion proteins and are also present close to the N terminus in other
v-SNAREs (37-39).

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Fig. 2.
Conserved structural elements of Vti1
proteins. A, -helical projection of a predicted
amphipathic -helix in Vti1p between amino acid residues 37 and 60 which resembles viral fusion peptides. Bulky hydrophobic amino acid
residues are printed in boldface letters and the hydrophobic
face is encircled. B, helical wheel projection of the
conserved predicted -helix. Uppercase letters, conserved
amino acids; lowercase letters, conserved in three Vti1
proteins; X, less conserved; boxes, bulky
hydrophobic amino acids; circles, charged amino acids ( D and E; + K and L);
small circles, charged amino acid present in three Vti1
proteins; arrows point to amino acid exchanges in the yeast
mutant Vti1 proteins encoded by vti1-1, vti1-2,
vti1-11, and vti1-12.
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An alignment of all Vti1 proteins revealed a domain of 75 amino acids
next to the transmembrane domain, which could be aligned without gaps
and shows blocks of high amino acid identity. Within this domain all
Vti1 proteins contain amino acid stretches that are predicted to form
amphipathic
-helical coiled coils with high probability (Fig. 1,
solid lines above the sequences) and lower probability
(dashed lines). Using the paircoil program (34) the
probability scores were: hVti1 0.69, mVti1 0.74, mVti1b 0.30, SpVti1
0.85, and Vti1 0.29. Coiled coil formation may require interaction with
other proteins as observed for SNAP-25, Sec9p, and the v-SNARE Snc1p
(35, 36). To obtain a reliable secondary structure prediction all Vti1
proteins were aligned using the Maxhom multiple sequence alignment
program (40), and the secondary structure of the alignment predicted
with the EMBL PHDsec program (41). The domain between amino acids 132 and 190 in the Vti1 alignment is predicted to be
-helical with a
very high probability. To visualize conserved features, the Vti1
alignment between amino acids 134 and 187 (Fig. 1A,
solid squares) was drawn as a helical wheel projection (Fig.
2B). Amino acid residues that were identical in all Vti1
proteins were depicted in uppercase letters. Residues that
are identical in three out of the four Vti1 proteins were drawn as
lowercase letters. hVti1 and mVti1 were treated as one protein due to their high degree of identity. Bulky hydrophobic amino
acids (L, M, V, I) are boxed, an empty box
represents a conserved bulky hydrophobic residue. Charged residues are
surrounded by a circle, a circle with a dash (-) represents
a conserved E or D, a circle with a plus (+) represents a conserved K
or R. X marks residues that are not conserved. The helical
wheel projection reveals that one face of the helix is highly
conserved. It consists of three leucines, an alanine, a glycine, and
three more leucines. Only in two cases is one of the leucines replaced
by an isoleucine. The neighboring face of the helix contains a
conserved hydrophobic and conserved charged amino acids, two identical
arginines, an acidic residue, and a less conserved basic residue. The
other parts of the helix are less well conserved.
As described recently, screens for the temperature-sensitive
vti1 mutants in yeast led to the identification of two
different classes of mutants (25). The mutants vti1-1 and
vti1-2 exhibit a block in transport of the vacuolar
hydrolase CPY from the late Golgi to the prevacuolar compartment at the
restrictive temperature. The mutant vti1-11 has a
temperature-sensitive growth defect and accumulates secretory proteins
in the ER and early Golgi compartment, in addition to a block in Golgi
to prevacuolar traffic. To determine which parts of Vti1p are involved
in these functions the vti1 mutant alleles were sequenced.
vti1-1 contains the amino acid exchanges E145K and G148R.
vti1-2 has the mutations S130P and I151T. 8 amino acid
exchanges were identified in vti1-11 (Y8R, K20R, H40R, N61S,
K73R, Q84R, E145G, and L155F). The construction of a hybrid protein
encoded by the plasmid pFvM108 revealed that yeast cells carrying a
protein with only the amino acid exchanges E145G and L155F exhibited
phenotypes identical to the original vti1-11 mutant (data
not shown). Therefore E145G and L155F are responsible for the
trafficking defect observed for vti1-11.
In our ongoing analysis of yeast Vti1p function we analyzed a new
VTI1 allele, vti1-12. The fate of newly
synthesized CPY was monitored in vti1-12 cells incubated at
22 °C or for 15 min at 37 °C by pulse-chase labeling followed by
CPY immunoprecipitation. Even at 22 °C vti1-12 cells
accumulated a significant proportion of CPY in the ER and early Golgi
forms (p1CPY) and the late Golgi form p2CPY intracellularly (42).
Hardly any vacuolar localized mature CPY (mCPY) was present
(Fig. 3, left panel,
I). These cells also secreted p2CPY (Fig. 3, left
panel, E). At 37 °C vti1-12 cells
exhibited a severe growth defect and accumulated an even higher
proportion of CPY in the ER and early Golgi form (Fig. 3, right
panel). These data indicate that the vti1-12 mutation causes a constitutive block of traffic from the late Golgi to the
vacuole and a temperature-sensitive block in traffic to the cis-Golgi
compartment. Vti1p encoded by vti1-12 carries the amino acid
exchanges A141S and Q158R. Therefore, vti1-12 and the other vti1 alleles characterized all contained mutations in
conserved amino acid residues in the predicted
-helical domain (Fig.
2B, arrows with mutant amino acid residues in
different vti1 alleles).

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Fig. 3.
CPY sorting in vti1-12
cells. CPY transport was assayed in vti1-12 cells
by pulse-chase labeling and CPY immunoprecipitations from cellular
extracts (I, intracellular) and medium (E,
extracellular). At 22 °C CPY did not reach the vacuole
(mCPY), but instead accumulated as the ER and early Golgi
p1CPY, and some late Golgi p2CPY accumulated and was secreted. At
37 °C after a 15-min preincubation, most CPY accumulated
intracellularly as p1CPY.
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With the exception of amino acid substitution S130P in
vti1-2, which could disrupt the structure of the
-helix,
all of the other mutations occur between residues 141 and 158. Interestingly, the same short stretch of Vti1p is affected in the
vti1 mutants independent of whether they affect the step
involving Pep12p (prevacuole) or Sed5p (cis-Golgi). It is striking that
strictly conserved amino acid residues in the conserved hydrophobic
face of the helix are mutated in three of the vti1 mutants.
This result suggests that this surface of Vti1p interacts with its
effector proteins in both late Golgi to prevacuolar transport, as well
as membrane traffic to the cis-Golgi.
Whereas the paircoil program predicted a coiled coil helix between
amino acids 119 and 153 of Vti1p with a low probability of 0.076, the
coiled coil probability dropped to 0 in the vti1-2, vti1-11 and vti1-12 mutant proteins. In contrast,
vti1-2 had a coiled coil probability of 0.13 between amino
acids 127 and 153. These structure predictions are consistent with the
possibility that the identified mutations could affect the structure of
this domain.
hVti1 Expression Suppresses vti1
Lethality--
As mentioned
above, hVti1 was identified as a suppressor that allowed
vti1
yeast cells to grow in the absence of Vti1p. To study the effects of hVti1 further, two expression plasmids were constructed. The coding regions for the complete hVti1 (codons 1-232)
and the coding region for the shorter version of hVti1 identified
through PCR amplification (codons 62-232) were amplified and cloned
into the multicopy yeast expression plasmid pVT100-U behind the yeast
ADH1 promoter. vti1
yeast strains with
VTI1 expressed under the GAL1 promoter were
transformed with both hVti1 expression constructs. Both strains were
able to grow when expression of yeast VTI1 was repressed,
and these cells could now lose the GAL1-VTI1 plasmid.
Doubling times were determined by measuring the optical density at 600 nm of cultures growing in logarithmic phase. The doubling times for
vti1
cells expressing hVti1 (1-232) and
vti1
cells expressing hVti1 (62-232) were almost
identical (3 h) and only slightly longer than the doubling time of wild type cells (2 h, 10 min) in rich medium (Fig.
4). These results demonstrate that hVti1
is able to perform the essential function of Vti1p and support growth
of vti1
cells quite well. These results also indicate
that the first 61 amino acids of hVti1 are not necessary for this
function.

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Fig. 4.
hVti1 expression suppresses vti1
lethality. Growth curves of wild type cells
(squares) and vti1 cells expressing hVti1
amino acids 62-232 (diamonds), or complete hVti1 (1-232, circles). The doubling times for vti1 cells
expressing hVti1 (62-232) and hVti1 (1-232) were essentially
identical (3 h) and only slightly longer than the doubling time of wild
type cells (2 h, 10 min). Cells were grown in rich medium at 30 °C
in logarithmic phase. Cell density was measured by optical density at
600 nm.
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Restoration of CPY Transport by Expression of hVti1--
hVti1
(62-232) was expressed in the temperature-sensitive mutants
vti1-1 and vti1-11 to assay its effect on the two
trafficking steps that have been shown to require Vti1p. At the
restrictive temperature, vti1-1 cells secreted and
accumulated the late Golgi form p2CPY (Fig.
5, top left panel,
hVti1 expression). This indicates a block in traffic from
the late Golgi to the prevacuolar compartment (25). Expression of hVti1
(62-232) led to the maturation and vacuolar localization
(mCPY) of a large fraction of the CPY (Fig. 5, top
left panel, + hVti1 expression). vti1-11
cells accumulated the ER and early Golgi form of CPY, p1CPY, at the
restrictive temperature (Fig. 5, top right panel). p2CPY,
which got past this first block, was secreted and did not reach the
vacuole. Expression of hVti1 (62-232) in vti1-11 cells
restored transport of CPY to the vacuole.

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Fig. 5.
Suppression of the CPY sorting defect by
hVti1 expression. CPY sorting was followed in vti1-1
cells (top left panel) and vti1-11 cells
(top right panel) in the absence ( , left) or presence (+, right) of hVti1 (amino acids 62-232) at
37 °C after a 15-min preincubation. hVti1 suppressed the block of
Golgi to vacuolar transport in vti1-1 cells, demonstrated by
the presence of intracellular mCPY. hVti1 also suppressed the cis-Golgi
traffic block in vti1-11 cells. In the absence of Vti1p
(vti1 , bottom panels) expression of hVti1
(amino acids 62-232) or the complete hVti1 (1-232) allowed sorting of
CPY to the vacuole with similar efficiency. Pulse-chase labeling was
performed at 30 °C in vti1 strains.
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To determine if hVti1 acts as a bypass suppressor or together with the
mutant Vti1p, hVti1 was expressed in vti1
cells. In vti1
cells expression of hVti1 (62-232) allowed a
significant fraction of CPY to reach the vacuole (Fig. 5, bottom
left panel), and some CPY was secreted. These data indicate that
hVti1 (62-232) can function in both Golgi to prevacuolar transport and
in traffic to the cis-Golgi in the complete absence of yeast Vti1p.
Expression of the complete hVti1 (1-232) in vti1
cells
(Fig. 5, bottom right panel) gave results similar to
expression of the shorter version hVti1 (62-232). This suggests that
the N-terminal 61 amino acids of hVti1 are not needed for function in
yeast. Maturation of CPY was slower in vti
cells than in
vti1-ts cells expressing hVti1 and a proteolytic
intermediate was observed at earlier time points (data not shown).
Rather than reflecting slower kinetics of vacuolar delivery this likely
indicates that the vacuole contains lower amounts of proteases in
vti1
cells expressing hVti1 than in wild type cells.
Suppression of the Invertase Sorting Defect by
hVti1--
Trafficking through the secretory pathway was further
studied using the secreted protein invertase (Fig.
6). Cells were shifted to 37 °C and
newly synthesized invertase was radioactively labeled during the pulse
and immunoprecipitated immediately or after a 30-min chase period.
Invertase received extensive Asn-linked carbohydrates in the ER during
the pulse period (Fig. 6, lane 9) and was converted to a
slow migrating, heterogeneous glycoprotein in the Golgi complex, and
then rapidly secreted in wild-type cells (Fig. 6, lanes 10 and 12) (33). In vti1-11 cells the core
glycosylated ER form of invertase accumulated intracellularly (Fig. 6,
lane 3, Inv. ER) and a severely underglycosylated
form was secreted after a 30-min chase at the restrictive temperature
(Fig. 6, lane 4) (25). No mature invertase was formed in
vti1-11 cells shifted to the high temperature. These data
indicate that ER to Golgi membrane traffic is drastically slowed down
and that the Golgi apparatus has lost the ability to glycosylate
invertase normally in vti1-11 cells at restrictive
temperature. In vti1-11 cells expressing hVti1 (62-232) the
core glycosylated ER form of invertase, which was found after the
pulse-labeling period (Fig. 6, lane 5), was absent after the
chase (lane 7). Instead, mature and underglycosylated invertase were found both within the cell and in the secreted fraction
(lanes 7 and 8). These data indicate that
expression of hVti1 suppressed the block in traffic to the cis-Golgi
compartment, which was observed in vti1-11 cells. A portion
of the invertase was not secreted and/or remained underglycosylated
after the 30-min chase period, suggesting that Golgi function was only
partially restored.

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Fig. 6.
Suppression of the invertase secretion defect
by hVti1 expression. vti1-11 cells, vti1-11 cells
expressing hVti1 (62-232), and wild type cells were grown at 24 °C
and preincubated (15 min) and labeled at 37 °C. Invertase
immunoprecipitations from intracellular (I) or extracellular
(E) fractions were performed directly after the 7-min pulse
or after a 30-min chase. In vti1-11 cells the ER form of
invertase accumulated and a severely under glycosylated form was
secreted (left panel). Expression of hVti1 in
vti1-11 cells led to the accumulation and secretion of
mature and underglycosylated invertase. The ER form of invertase was
absent (middle panel). In wild type cells, mature invertase
was secreted (right panel).
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DISCUSSION |
Identification of the Human Vti1 Homolog--
A human homolog
(hVti1) of the yeast Vti1p was identified by functional
complementation. A mouse homolog, mVti1, which is 93% identical to
hVti1 over the full length of the protein, was assembled from data base
EST sequences. The homology did not extend upstream of the putative
initiating methionine, indicating that the proteins are in fact 232 amino acids long. In addition, two clones amplified by PCR as well as a
human EST (AA056932) found in the data base encode hVti1 starting from
methionine 62 but contain three different sequences upstream. These
clones could represent cloning artifacts. However, it is clear that
both the full length and the short version are functional in yeast.
Therefore, it is also possible that the different 5
ends of the
mRNA could be derived through alternative splicing and that both
proteins could exist in humans. This would require that the spliced
RNAs are of similar length because only one mRNA band was
identified in Northern blots of RNA from human tissues.
Two mouse proteins, mVti1 and mVti1b, were assembled from the EST data
base and they exhibit similar homology to yeast Vti1p (27-35%).
Surprisingly, mVti1 and mVti1b share only 31% amino acid identity.
Redundant proteins are common in mammals, but are usually much more
similar to each other. Two different mammalian Sec22p homologs have
been identified. Although from related species (rat and hamster),
rSec22 and ERS-24 share only 35% amino acid identity (9, 10). It
remains unclear whether these proteins function in the same or in
different transport steps. The tagged and overexpressed rSec22 protein
localizes to the ER, whereas ERS-24 localizes to the ER and Golgi.
In vitro ERS-24 was found in a complex with Sed5p. The amino
acid sequences of the redundant v-SNAREs synaptobrevin1/VAMP1 and
synaptobrevin2/VAMP2 are 77% identical (43, 44). In contrast,
cellubrevin, which acts in a similar but distinct membrane traffic
step, has 59% amino acid identity with the other mammalian
synaptobrevins (45). It therefore seems more likely that mVti1 and
mVti1b function in different trafficking steps.
Implication for the Structure of v-SNAREs--
In an effort to
determine which domains of v-SNAREs and t-SNAREs interact, several
groups have characterized different SNARE fragments in in
vitro binding assays. Syntaxin 1 interacts through the putative
coiled coil helix 3 (amino acids 190-240) with both synaptobrevin and
SNAP-25 (46, 47). SNAP-25 binds to syntaxin through the N-terminal
predicted coiled coil region (amino acids 1-100). SNAP-25 requires its
N- and C-terminal coiled coil regions for binding to synaptobrevin.
These data suggest that t-SNAREs interact via coiled coil domains with
v-SNAREs. Isolated SNAP-25 has a very low
-helical content, but in a
SNAP-25 syntaxin complex the
-helicity is increased dramatically
(36). The related yeast SNAREs behave in a similar way. The isolated
v-SNARE Snc1p and the t-SNARE Sec9p are largely unstructured (35). The
formation of
-helices is induced in a complex of Snc1p, Sec9p and
Sso1p and thermal stability is increased. These data indicate that
SNAREs exist in different conformations and that coiled coil structures are induced by complex formation. The transition between unfolded and
folded states may play an important role in docking and fusion. In vitro binding assays have not been particularly
successful to narrow down the interacting domains of v-SNAREs.
Synaptobrevin2 requires most of the cytosolic domain (amino acids
27-96) for its interaction with syntaxin (48). In a functional assay,
amino acids 31-80 of synaptobrevin2 were required to restore
exocytosis (49).
Using sequence analysis of temperature-sensitive vti1
mutants we have been able to narrow down the functional domain of
Vti1p. We analyzed two mutants defective in Golgi to prevacuolar
transport (mediated by the prevacuolar t-SNAREs Pep12p), and two
mutants with additional defects in trafficking to the cis-Golgi, which involves the cis-Golgi t-SNARE Sed5p. Amino acid exchanges in both
classes of mutants cluster in a very narrow region between amino acid
residues 130 and 158. This area is predicted to be
-helical and to
fold into a coiled coil structure with low probability. It is
immediately adjacent to the domain that is predicted to form a coiled
coil structure with higher probability (amino acids 159-188). In
analogy to Snc1p, Sec9p and SNAP-25, folding may require the presence
of a t-SNARE.
Functional Complementation--
hVti1 was capable of replacing
Vti1p in both Golgi to prevacuolar transport and traffic to the
cis-Golgi compartment. The first 61 amino acid of hVti1 were not needed
for these functions in yeast. This means that hVti1 does not need most
of the conserved predicted amphipathic
-helix between amino acid
residues 43 and 69. hVti1 contains an additional amphipathic domain
(amino acid residues 77-98), and these domains may be functionally
redundant. The ability of hVti1 to functionally complement in yeast is
quite surprising given the low degree of amino acid identity between the human and the yeast proteins (29%). Clearly, the protein's structure and key functional residues must be conserved. The most conserved feature among the different Vti1-like proteins is a hydrophobic face in a predicted
-helix between amino acids 134 and
181. All the amino acid exchanges identified in the mutant yeast Vti1p
proteins are in evolutionary conserved residues in this region and on
the same side of the predicted
-helix. Therefore, it seems very
likely that this hydrophobic face, which is lined by charged residues,
represents the part of Vti1p that interacts with its effector proteins,
possibly with both Pep12p and Sed5p.
We thank N. J. Bryant and W. Voos for
critical reading of the manuscript. We also thank J. Colicelli for
providing the human glioblastoma cDNA library in the pADANS yeast
expression vector.