Biochemical and Genetic Evidence for the Involvement of Yeast
Ypt6-GTPase in Protein Retrieval to Different Golgi Compartments*
Zongli
Luo and
Dieter
Gallwitz
From the Max Planck Institute for Biophysical Chemistry, Department
of Molecular Genetics, D-37070 Göttingen, Germany
Received for publication, September 6, 2002, and in revised form, October 18, 2002
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ABSTRACT |
Yeast Ypt6p, the homologue of the mammalian Rab6
GTPase, is not essential for cell viability. Based on previous studies
with ypt6 deletion mutants, a regulatory role of the GTPase
either in protein retrieval to the trans-Golgi network or
in forward transport between the endoplasmic reticulum (ER) and early
Golgi compartments was proposed. To assess better the primary role(s) of Ypt6p, temperature-sensitive ypt6 mutants were generated
and analyzed biochemically and genetically. Defects in
N-glycosylation of proteins passing the Golgi and of
Golgi-resident glycosyltransferases as well as protein sorting defects
in the trans-Golgi were recorded shortly after functional
loss of Ypt6p. ER-to-Golgi transport and protein secretion were delayed
but not interrupted. Mis-sorting of the vesicular SNARE Sec22p
to the late Golgi was also observed. Combination of the
ypt6-2 mutant allele with a number of mutants in forward
and retrograde transport between ER, Golgi, and endosomes led to
synthetic negative growth defects. The results obtained indicate that
Ypt6p acts in endosome-to-Golgi, in intra-Golgi retrograde
transport, and possibly also in Golgi-to-ER trafficking.
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INTRODUCTION |
Intercompartmental transport of proteins in secretion and
endocytosis is affected by a variety of vesicle populations, each having its own biochemical identity. Generation of transport vesicles at different donor compartments and their fusion with defined target
membranes follow general principles and require protein machines made
up of related and evolutionarily conserved proteins that are, however,
specific for each transport step (1, 2). Among the proteins that confer
specificity and directionality to vesicular trafficking are Ras-like
GTPases of the Ypt/Rab family (3-6). In the yeast Saccharomyces
cerevisiae, this GTPase family has 11 members of which only those
that act in forward transport through the secretory pathway (Ypt1p,
Sec4p, and the pair of apparently redundant Ypt31/Ypt32-GTPases) are
essential for cell viability (3). Of the nonessential ones, Ypt6p, the
homologue of the mammalian Rab6 GTPases, has been implicated in having
a role in the retrieval of proteins from endosomes to the
trans-Golgi network (7-10) or in anterograde (11, 12) and
retrograde (9) Golgi transport.
The divergent views on Ypt6p function were based on the results of the
initial studies (7, 8, 11, 12) using ypt6 deletion or
truncation mutants. Such mutants were found to be growth-inhibited at
elevated temperature, to partially mis-sort proteins in the
trans-Golgi network already at permissive temperature, and
to moderately interfere with protein secretion at nonpermissive conditions (7, 11). Temperature sensitivity of ypt6 deletion mutants allowed us to isolate multicopy suppressors of which one, SYS1 (7), could also rescue another protein mis-sorting
mutant, ric
(9), later shown to be defective in the
nucleotide exchange factor for Ypt6p (13). Cells lacking Ypt6p
accumulate transport vesicles (8) of which at least a fraction may be
derived from endosomes and destined to fuse with late Golgi membranes
(10). On the other hand, temperature-sensitive growth of
ypt6 deletion mutants can be overcome efficiently by raising
the intracellular level of Ypt1p (12), the GTPase required for
ER1-to-Golgi (14) and early
Golgi transport (15). Furthermore, partial suppression of growth
defects of ypt6 and ric1 null mutants by high
expression of a variety of genes that are known to act in either
forward or retrograde Golgi transport have led to the assumption that
Ypt6p might participate in regulating more than one transport step (9).
This would in fact mirror the situation in mammalian cells where the
homologue of yeast Ypt6p, Rab6, initially thought to function only in
intra-Golgi transport (16), is also required for recycling
of proteins between endosomes and the TGN (17). The notable difference,
however, is that although yeast has only one form of Ypt6p, mammalian
cells are endowed with two Rab6 isoforms that apparently act
sequentially in transport from endosomes to the early Golgi (17) and
even to the ER (18).
As previous studies with ypt6 deletion mutants have the
inherent problem that adaptive changes in the physiology of such cells might obscure the true lesions caused by functional loss of the GTPase,
we sought to investigate Ypt6p function with the help of conditional
mutants. The data obtained from genetic interactions and from
experiments following the kinetics of protein transport and sorting as
well as the state of protein glycosylation at permissive and
nonpermissive conditions are best explained by Ypt6p acting both in
recycling of proteins from endosomes to the Golgi and from late to
early Golgi compartments.
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EXPERIMENTAL PROCEDURES |
Yeast Strains, Genetic Methods, and Plasmids--
Yeast strains
used in this study are listed in Table I.
Yeast transformations, mating, sporulation, and tetrad analyses were performed using standard techniques (19, 20). c-Myc epitope tagging of
ORFs was performed as described previously (21). To produce
pKS-YPT6-URA3 for site-directed mutagenesis, the 1.4-kb XbaI
YPT6 gene fragment from pRS315-YPT6 was inserted into the XbaI site of pBluescript II KS+ (Stratagene).
The HindIII URA3 gene fragment from YEp24 (New England Biolabs) was inserted into the StuI site of
YPT6 created 22 bp behind the stop codon. To create
pYX242-SEC35, the coding sequence of SEC35 gene with a newly
created NcoI site at the 5' end and an XhoI site
at the 3' end was amplified by PCR from yeast genomic DNA using
Pfu DNA polymerase (Stratagene). This fragment was inserted
into the NcoI-XhoI sites of pYX242 (R&D Systems). Plasmids pRS315-YPT6 and pRS325-SYS1 were constructed as described previously (7); pRS326-YPT1 was from R. Peng (this laboratory), and
pWB-Acyc
(PCYC1-SEC22-myc-
factor,
CEN, URA3) was from H. D. Schmitt (this
laboratory).
Site-directed Mutagenesis and Gene
Disruption--
YPT6 mutant genes encoding the GTPase with
either K125N, G139D, or A143D substitution were generated by
site-directed mutagenesis using QuickChangeTM mutagenesis
kit (Stratagene) on the plasmid pKS-YPT6-URA3. After mutation, an
XbaI fragment of the mutated YPT6 gene with the
adjacent URA3 gene as selection marker was integrated into
the YPT6 locus of a haploid strain, and mutants were
selected on SD-Ura plates. Chromosomal mutations were verified by
sequencing the PCR products generated from genomic DNA of selected
ypt6 mutants. Disruption of genes was performed by PCR-based
replacement using the HIS3 gene amplified from plasmid
pRS303 (22) to replace the respective ORFs as described (23).
Protein Extraction, PNGase F Treatment, and
Immunoblotting--
Yeast cells were grown at 25 °C to mid-log
phase. Ten A600 units of cells were
collected before and after incubation at 37 or 39 °C for different
times, resuspended in 150 µl of SDS sample buffer containing
proteinase inhibitors, and lysed by vortexing for 10 min at 4 °C
with glass beads. For PNGase F digestion, cells were collected,
spheroplasted, and gently lysed in HEPES buffer. The lysates were then
centrifuged at 100,000 × g for 2 h at 4 °C;
the pellets were resolved in 0.5% SDS buffer containing 1%
-mercaptoethanol, and digestions were done according to the
protocols advertised by the supplier (New England Biolabs). For
immunoblotting, proteins were resolved by SDS-PAGE, transferred to
nitrocellulose membranes, and probed with polyclonal antibodies against
c-Myc epitope (Santa Cruz Biotechnology), CPY, ALP, or chitinase.
Cell Labeling, Immunoprecipitation, and Invertase
Assay--
Cell labeling and immunoprecipitations were performed as
described previously (24) with the following modifications. Cells were
grown in synthetic minimal medium containing 2% glucose (SMM) (25) to
mid-log phase and labeled with Tran35S-label (ICN) in
either SD medium supplemented with the required amino acids or in SMM
containing 1 M sorbitol and 1 mg/ml ovalbumin. Labeling in
SMM was terminated by adding 10 mM NaN3/NaF and
in SD medium by 2× spheroplast buffer (50 mM Tris, pH 7.5, 2 M sorbitol, 20 mM NaN3/NaF, 20 mM dithiothreitol). Spheroplasts were made by adding 10 µg of zymolase 100-T (Seikagaku Kogyo) per 1 A600 unit of cells and incubating for 45 min at 30 °C. Secretion of proteins into the medium was assayed as
described (26). Invertase activity staining was carried out as
described previously (27).
Sucrose Gradient Fractionation--
Yeast cells were
grown at 25 °C to mid-log phase and then shifted to 37 °C for
1 h. 150 A600 units of cells were
collected. Lysates were prepared and subjected to sucrose gradient
centrifugation as described (28). Thirteen fractions were collected
manually from the top of the gradient and mixed with one-fifth volume
of 6× SDS sample buffer and incubated at 95 °C for 10 min prior to SDS-PAGE. Immunoblots were performed using specific antibodies against
Kar2p, Emp47p, CPY, ALP, Anp1p, and the c-Myc epitope.
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RESULTS |
Growth Phenotype of ypt6 Temperature-sensitive Mutants--
Ypt6p
is not required to sustain cell growth and multiplication. Therefore,
previous studies (7, 8, 11, 12) on the functional role of Ypt6p were
performed with ypt6 deletion or truncation mutants which
are, however, sensitive to growth at elevated temperature. Since
mutants lacking a given protein are sometimes able to adapt to such a
situation by activating a by-path for example, we sought to investigate
Ypt6p function in conditional mutants. By analogy with other
heat-sensitive Ypt proteins, three single amino acid substitutions were
introduced into the conserved (NKXD) sequence, known to be
involved in nucleotide binding, and into the helical region following
this sequence (Fig. 1A). Each of the three mutant alleles, ypt6-1 (expressing
Ypt6(K125N)p), ypt6-2 (expressing Ypt6(G139D)p), and
ypt6-3 (expressing Ypt6(A143D)p), conferred heat sensitivity
to a varying extent on haploid cells grown in rich media (Fig.
1B). ypt6-2 cells exhibited the most severe
growth defect. Like ypt6
cells of the same genetic
background, this mutant grew slowly at 35 °C and failed to
proliferate at 37 °C. The ypt6-1 mutant grew well at
37 °C but extremely slowly at 39 °C, whereas growth of
ypt6-3 cells slowed down at 37 °C and completely failed
at 39 °C. Compared with wild type, all ypt6 mutants
exhibited somewhat reduced growth at 14 °C.

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Fig. 1.
Generation and growth phenotype of
ypt6 mutants. A, location of single
amino acid substitutions in Ypt6p of three different mutants.
G1-5, conserved GTPase domains; C, conserved
C-terminal cysteine residue. B, 3.0 µl of 10-fold diluted
log-phase cultures, starting with 3 × 106 cells/ml,
were spotted onto YPD or YPDS (YPD plus 1 M sorbitol)
plates and incubated for 3 days at the indicated temperatures.
Incubation at 14 °C was for 10 days. C, WT (MSUC-3D),
ypt6-2 (ZLY-2), and ypt6 (ZLY4) strains were
grown at 25 °C to saturation. 3.0 µl of 10-fold diluted cultures,
starting with 8 × 106 cells/ml, were spotted onto
designated plates and incubated at the indicated temperatures for 2 days. CFW, calcofluor white (Fluorescent Brightener 28;
Sigma)
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The growth defects of ypt6 mutants at high temperature were
partially rescued by the addition of 1 M sorbitol, a
phenotype described for mutants with a defective cell wall (29).
Therefore, we tested whether ypt6 mutants are sensitive to
SDS and calcofluor white (CFW), agents affecting cell wall integrity
and being more toxic to mutants with a defective cell wall. Compared
with the wild type strain, ypt6-2 and ypt6 null
mutants were more sensitive to 0.005% SDS and 0.005% CFW (Fig.
1C), indicating a cell wall defect in these mutants.
Protein Trafficking Defects in ypt6 Mutants--
We selected
vacuolar carboxypeptidase Y (CPY), alkaline phosphatase (ALP), and the
secreted invertase as markers to test whether the intracellular vesicle
transport was affected in the ypt6 mutants. These three
proteins are N-linked glycoproteins that undergo core glycosylation in the ER and outer chain elongation in the Golgi before
being transported to their final destination via different routes. CPY
is transported via endosomes to the lumen of the vacuole, where it is
processed to the mature form by proteolysis (30). ALP is transported
directly to the membrane of the vacuole without passing through
endosomes (31) and is also cleaved by proteolysis in the vacuole.
Invertase is rapidly transported to the periplasmic space (32). By
monitoring the molecular size and/or localization, it can be determined
which step of transport is affected in the mutants. At steady state,
accumulation of core-glycosylated CPY (p1-form) was detected in
ypt6-1, ypt6-2, and ypt6-3 when shifted to the
nonpermissive temperature (37 or 39 °C) for 1 and 2 h (Fig. 2A). Further kinetic analysis
of CPY by pulse-chase experiments showed that at permissive temperature
(25 or 30 °C), ypt6 mutants exhibited somewhat delayed
maturation of the enzyme and a partial mis-sorting of the
Golgi-glycosylated p2 form to the extracellular space. At nonpermissive
temperature, ypt6 mutants exhibited significantly reduced
CPY transport kinetics. However, defects in the Golgi-specific glycosylation were demonstrated by the presence of a smear between p2CPY and p1CPY (compare wild type and mutants in Fig. 2, B
and C). This is best seen in the experiment shown in Fig.
2C in which cells initially grown at 25 °C were taken up
in SMM containing 1 M sorbitol for cell stabilization,
preincubated at either 25 or 37 °C for 10 to 60 min, and then
radioactively labeled for 10 min at the respective temperature.
Although the extent of CPY labeling decreased with the length of time
the cells were incubated in the hypertonic medium, mis-sorting of a
significant fraction of the Golgi-glycosylated vacuolar enzyme was
evident already 10 min after shift of ypt6-2 cells to the
nonpermissive temperature, and the p2 form of CPY decreased in size at
later time points. At 60 min of preincubation at 37 °C, p2-CPY of
the ypt6-2 mutant cells, in contrast to wild type cells, was
detectable only as a smear above the sharp band of the ER
core-glycosylated enzyme, clearly demonstrating a severe glycosylation
defect in the Golgi. The defects observed in ypt6-2 mutants
were more severe than those in ypt6-1 and ypt6-3
cells, consistent with the growth phenotype. As shown in Fig.
3A, pro-ALP was also
accumulated in ypt6 mutants at steady state upon shift to
nonpermissive temperature. Further analysis by pulse-chase experiments
indicated that at 25 °C, maturation of ALP was normal in
ypt6-2 mutant cells. However, at 37 °C, pro-ALP was
accumulated and, in gels, migrated progressively slower at chase times
from 0 to 30 min (Fig. 3B), suggesting ongoing glycosylation in the Golgi.

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Fig. 2.
CPY transport analysis in ypt6
mutants. A, Western blot analysis of CPY with
total protein extracts. WT (MSUC-3D), ypt6-1 (ZLY1),
ypt6-2 (ZLY2), ypt6-3 (ZLY3),
ypt1A136D (YXY136), and
ypt32A141D/ypt31 (YLX15) cells were
grown at 25 °C to mid-log phase and shifted to 37 or 39 °C for 0, 1, or 2 h before lysis. After SDS-PAGE, transfer to nitrocellulose
filters and treatment with anti-CPY antibody, CPY was visualized by
fluorography. B, pulse-chase analysis with WT (MSUC-3D) and
ypt6 mutants (ZLY1, ZLY2, and ZLY3). Cells were either
incubated at permissive temperature or shifted to nonpermissive
temperature for 30 min in SD medium containing required amino acids and
1 mg/ml of ovalbumin, labeled for 7 min with Tran35S-label,
and then chased with cold methionine and cysteine for the indicated
times. The cells were then spheroplasted, and intracellular
(I) and extracellular (E) fractions were
collected. CPY was immunoprecipitated from both fractions, resolved by
SDS-PAGE, and visualized by autoradiography. Note that mis-sorting of
CPY occurred already at permissive temperatures. C, WT and
ypt6-2 mutant cells were preincubated in SMM containing 1 M sorbitol and 1 mg/ml ovalbumin at the indicated times and
temperatures, labeled with Tran35S-label for 10 min, and
chased for 30 min. Newly synthesized CPY was identified as described in
B.
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Fig. 3.
ALP maturation in ypt6
mutants. A, Western blot analysis of ALP with
total cellular protein. The same extracts used in the experiment of
Fig. 2A were analyzed with anti-ALP antibody. B,
pulse-chase analysis with WT (MSUC-3D) and ypt6-2 (ZLY2)
mutants. Cells were grown and subjected to pulse-chase analysis as
described in the legend to Fig. 2B. ALP was
immunoprecipitated from cell lysates, resolved by SDS-PAGE, and
visualized by autoradiography. * denotes an unrelated protein band that
does not change electrophoretic behavior after PNGase F
treatment.
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Invertase was analyzed both in the intracellular and extracellular
fraction by activity staining in non-denaturing polyacrylamide gels. At
permissive temperature, the secretion of highly glycosylated invertase
was normal in ypt6 mutants as compared with wild type cells.
At nonpermissive temperature, ypt6-1 and ypt6-3
mutants showed slight defects in the secretion and glycosylation of the enzyme. Importantly, at 37 °C invertase in ypt6-2 mutant
cells was clearly underglycosylated, and somewhat less than half of the
enzyme was accumulated inside the cell. As controls for ER-to-Golgi transport mutants, processing and secretion of invertase was
investigated in sec18-1 and ypt1A136D
strains at nonpermissive conditions. In these mutants, the enzyme was
nearly completely trapped inside the cells in ER-glycosylated forms
(Fig. 4A). To analyze further
whether Ypt6p is involved in secretion, we assayed for the export of
proteins into the growth medium (24, 26). Wild type and mutant cells
were preincubated at 37 °C for 30 min and then subjected to
pulse-chase at 37 °C. Cells were removed by centrifugation, and the
proteins in the medium were precipitated with trichloroacetic acid,
resolved by SDS-PAGE, and identified by autoradiography. As shown in
Fig. 4B, no protein band was detected in the medium of
sec18-1 cells, and only very faint bands were seen in the
medium of ypt1A136D cells, indicating the known
secretion block in these two mutants. However, the same set of secreted
proteins, including the most prominent band at 130-150 kDa that
represents HSP150 (24, 26), was visible in the medium of wild type,
ypt6-2, and ret2-1 cells, the latter being
defective in retrograde transport from the Golgi to the ER (33).
Comparing the band intensities, it appears that secretion in
ypt6-2 mutants was somewhat reduced.

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Fig. 4.
Secretion and Sec22p recycling analysis of
ypt6 mutants. A, WT (MSUC-3D),
ypt6-1 (ZLY1), ypt6-2 (ZLY2), ypt6-3
(ZLY3), sec18-1 (YTX50), and ypt1A136D
(YXY136) strains were grown at 25 °C to mid-log phase; and cells
were washed and resuspended in 0.1% glucose-containing medium to
induce secreted invertase at 25 or 37 °C for 1 h. Intracellular
(I) and extracellular (E) fractions were
separated, and invertase was analyzed by activity staining in
non-denaturing acrylamide gels. B, WT (MSUC-3D),
ypt6-2 (ZLY2), ypt1A136D (YXY136),
ret2-1 (PC130), and sec18-1 (YTX50) strains were
preincubated for 30 min at 37 °C, labeled for 10 min with
Tran35S-label, and then chased for 30 min. Proteins
secreted into the medium were precipitated with trichloroacetic acid
and analyzed by SDS-PAGE and autoradiography. C, recycling
defect of Sec22p. WT (MSUC-3D), ypt6-2 (ZLY2-K), and
ret1-1 (PC70) cells harboring the plasmid pWB-Acyc
encoding Sec22p-myc- factor ( ) fusion protein were grown
overnight at 25 °C to mid-log phase in SC-Ura medium, and then half
of the cultures was shifted to 37 °C for 1 h. Total proteins
were extracted from the cells before and after 1 h of treatment at
37 °C and analyzed by immunoblot using anti-Sec22p antibody or
anti-c-Myc antibody. Sec22p-myc, the Kex2 protease cleaved products
from Sec22p-myc- fusion protein; Sec22p, wild type v-SNARE. Note
that in the ret1-1 mutant the fusion protein is almost
completely cleaved.
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We next examined the recycling of the v-SNARE Sec22p in
ypt6-2 mutant cells. Sec22p is known to cycle between the
Golgi and the ER as well as between Golgi compartments. In wild type
cells, only a small fraction of the v-SNARE reaches the late Golgi, but in mutants defective in retrograde transport of ER resident proteins, Sec22p is easily mis-sorted to this compartment (34, 35) in which the
Kex2 protease resides. For this study, we took advantage of an
Sec22-
factor fusion protein with a Kex2p cleavage site in its
linker region and a c-Myc epitope tag adjacent to Sec22p. Cleavage of
the fusion protein signals arrival in the late Golgi (34). As shown in
Fig. 4C, the fraction of the fusion protein cleaved was
significantly higher in ypt6-2 mutant cells compared with
wild type cells independent of the temperature at which the experiment
was conducted. This clearly indicates a recycling defect in
Ypt6p-defective cells that could involve retrograde Golgi and/or Golgi-to-ER trafficking.
Glycosylation Defects of Golgi-localized Glycosyltransferases in
ypt6-2 Mutants--
The glycosylation defects of CPY and invertase in
ypt6 mutants prompted us to examine whether the defects
resulted from instability of glycosyltransferases. In yeast cells,
outer chain elongation of glycoproteins is catalyzed by
glycosyltransferases in the Golgi. Among them, Anp1p, Mnn9p, and Van1p
have been reported to recycle between the ER and the Golgi and are
subject to mislocalization and to degradation in the vacuole when
retrograde transport to the ER is blocked (36). We integrated c-Myc
epitope-tagged versions of ANP1, MNN9, and VAN1
into the genome of wild type and ypt6-2 strains and followed
the fate of the tagged proteins by immunoblot analysis. As shown in
Fig. 5A, a shift of cells to
37 °C for 2 h did not result in a significant reduction of
Anp1p-myc, Mnn9p-myc, and Van1p-myc in ypt6-2 compared with
wild type cells. Also no redistribution of Mnn9p-myc and Anp1p was
detected in ypt6-2 mutant cells in sucrose gradient analysis
(Fig. 6D), suggesting other reason(s) for the glycosylation defects observed. Likewise, there was
no decrease of the Golgi protein Emp47p following shift of ypt6-2 cells to nonpermissive temperature (Fig.
5A).

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Fig. 5.
Western blot analysis of
glycosyltransferases, Emp47p, and chitinase in
ypt6-2. A, stability of Myc-tagged
Anp1p, Mnn9p, and Van1p. WT and ypt6-2 strains expressing
c-Myc-tagged Anp1p (ZLY354, ZLY359), Mnn9p (ZLY353, ZLY358), and Van1p
(ZLY355, ZLY360) were grown at 25 °C to mid-log phase and shifted to
37 °C for 0, 1, or 2 h before lysis. Total proteins were
analyzed by immunoblot using anti-c-Myc or anti-Emp47p antibodies.
B, samples were prepared from WT, ypt6-2, and
sec23-1 strains expressing c-Myc-tagged Och1p (ZLY351,
ZLY356, and ZLY184) and Mnn1p (ZLY352, ZLY357, and ZLY185) before or
after shift to 37 °C for 1 h. Total proteins were subjected to
immunoblot analysis using anti-c-Myc and anti-chitinase antibodies.
p, core-glycosylated form; m, mature form.
C, endoglycosidase treatment. WT and ypt6-2 cells
expressing c-Myc-tagged Och1p (ZLY351, ZLY356) or Mnn1p (ZLY352,
ZLY357) were shifted to 37 °C for 1 h, and membrane proteins
were extracted, treated with PNGase F, and visualized on Western blots
using anti-c-Myc antibody.
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Fig. 6.
Distribution of proteins in sucrose gradients
of ypt6-2 lysates. WT (A),
ypt6-2 (B), and sec23-1 (C)
strains expressing Myc-tagged Och1p (ZLY351, ZLY356, and ZLY184) were
grown at 25 °C to mid-log phase and shifted to 37 °C for 1 h. Spheroplasts were generated and lysed gently. Lysates were
fractionated by sucrose gradient (18-60%) centrifugation and assayed
for the distribution of Och1p-myc, Emp47p, Kar2p, CPY, and ALP by
immunoblot analysis using specific antibodies. Relative levels of
proteins on immunoblots were quantified using a Lumi-Imager.
D, WT and ypt6-2 strains expressing Mnn9p-myc
(ZLY353 and ZLY358) were used to make lysates and subjected to
fractionation and immunoblot analysis as described above.
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Two other important glycosyltransferases are Och1p and Mnn1p. Och1p is
the first enzyme required for outer chain elongation of
N-linked glycoproteins in the cis-Golgi (37). It
does not recycle back to the ER (36) but was reported to recycle
between the TGN and the cis-Golgi (38). Mnn1p is responsible
for the termination of both N- and O-linked
glycosylation in the Golgi. It resides in two discrete Golgi
compartments and may cycle between the earlier compartments and the TGN
(39). To investigate whether Ypt6p might be involved in the recycling
of Och1p and Mnn1p, we followed their fate by a similar strategy as
described above. Two bands of Och1p-myc were detected in wild type,
ypt6-2, and sec23-1 cells at 25 °C. The upper
broad band could in fact contain three bands, because Och1p was
detected previously in four forms of 58-66 kDa (37). When cells were
shifted to 37 °C for 1 h, a band about 1-2 kDa smaller than
the upper broad band was accumulated both in ypt6-2 and in
sec23-1 cells (Fig. 5B). Because in
sec23-1 cells, the exit of proteins from the ER is blocked,
the additional band accumulating in the mutant cells was likely to
represent the core-glycosylated form of Och1p-myc. This was confirmed
by PNGase digestion, which removes all N-linked sugar
chains from the protein (Fig. 5C). The core-glycosylated
form of Mnn1p-myc was also detected in ypt6-2 and
sec23-1 mutant cells after a shift to 37 °C (Fig. 5,
B and C).
We also examined the extensively O-glycosylated protein
chitinase that is located in the cell wall. Although the results of this investigation were somewhat variable, chitinase in
ypt6-2 mutant cells had no significantly altered
electrophoretic mobility compared with wild type cells. However,
accumulation of the ER form was seen only in sec23-1 but not
in ypt6-2 mutant cells (Fig. 5B), again arguing
against an inhibition of ER-to-Golgi transport in the ypt6-2
mutant. These results indicate that in ypt6-2 mutant cells,
N-glycosylation in the Golgi is severely impaired, but O-glycosylation apparently is not.
Subcellular Localization of Core-glycosylated Forms of N-Linked
Glycoproteins in ypt6-2 Mutants--
We have shown that at steady
state, the core-glycosylated forms of CPY, ALP, Och1p-myc, and
Mnn1p-myc are accumulated in ypt6 mutants at nonpermissive
temperature. This accumulation may be the result of either a block in
ER-to-Golgi transport as proposed previously (11) or may be caused by
the lack of outer chain elongation of these glycoproteins. To
distinguish between these possibilities, we performed a subcellular
fractionation analysis, and we examined the localization of the
underglycosylated and/or core-glycosylated proteins. Cell lysates were
prepared from wild type, ypt6-2, and sec23-1
strains carrying a Myc-tagged version of OCH1. After
preincubation at 37 °C for 1 h, cells were collected, and
lysates were prepared and subjected to sucrose gradient centrifugation. Following gel electrophoretic separation of proteins in different gradient fractions, immunoblot analyses were performed with specific antibodies against the Golgi membrane protein Emp47p, the soluble ER
protein Kar2p, the vacuolar hydrolases CPY and ALP, and against the
c-Myc epitope for the identification of Och1p-myc. Band intensities of
immunoblots were quantified using a Lumi-Imager. As shown in Fig.
6A, in wild type cells, Kar2p was enriched in the three
bottom fractions of highest sucrose density which contain ER and plasma membranes. The mature forms of CPY and ALP were in the three upper fractions of the gradient containing vacuoles and soluble proteins. Only very small fractions of core-glycosylated proforms were found. Emp47p that recycles between the Golgi and the ER (28) was distributed in two peaks between fractions 4 and 13 with the main peak in fractions
8-10 in most of the experiments. The peak of Och1p-myc was detected
primarily in the latter fraction. In sec23-1 cells with an
inhibition of ER export, core-glycosylated proteins were mostly trapped
in the ER as expected. This also applied to the proforms of CPY, ALP,
and Och1p-myc that were particularly enriched in the three bottom
fractions of the gradient (Fig. 6C). In addition, the peak
of Emp47p was shifted to the ER membrane-containing region of the
gradient. However, in ypt6-2 mutant cells, most of the core-
and underglycosylated forms of CPY, ALP, and Och1p-myc were distributed
between fractions 6 and 13, with a peak around fractions 8-10, like
the Golgi marker Emp47p (Fig. 6, B and D) and the
Golgi-localized glycosyltransferases Mnn9p-myc and Anp1p (Fig.
6D). These results clearly indicate that most of the
core-glycosylated proteins reached the Golgi cisternae in
ypt6-2 cells at nonpermissive temperature, arguing against a
severe ER-to-Golgi trafficking defect.
We repeatedly observed that in the gradients used, Emp47p from cells
grown at 37 °C for 1 h appeared in two peaks in contrast to
ypt6-2 cells where Emp47p was mostly confined to one peak
coinciding with the Golgi glycosyltransferases (Fig. 6, A,
B and D). As in the ypt6-2 cells there
is no indication for mis-sorting of Emp47p to the vacuole that would
have been resulted in a decrease of the intracellular level of the
protein (28), and this finding is probably due to a disturbance of
Golgi homeostasis in cells defective in Ypt6p function.
Genetic Interactions of ypt6-2 and Mutants Defective in Transport
between ER and Golgi or Endosomes and the TGN--
Previous studies
(7, 9, 12) had shown that overexpression of either Sys proteins, Ypt1p,
Gos1p, or Ykt6p suppressed temperature-sensitive growth defects of
ypt6 null mutants. In the present study we could show that
high intracellular levels of Sys1p and of Ypt1p efficiently rescued
also the ypt6-2 mutant from growth inhibition at 37 °C
(data not shown). We additionally searched for synthetic growth defects
between ypt6-2 and other mutants defective in either
ER-to-Golgi transport (sec12-4, sec23-1, sec24-11,
ypt1A136D, yip1-2, sly1ts,
sed5-1, bet1-1, uso1-1, sec18-1, and sec35-1), the
Golgi-to-ER recycling (ret1-1, ret2-1, ret3-1, sec20-1,
sec21-1, and sec27-1), intra- and post-Golgi transport
(sec7-1, ypt32A141D/ypt31
, and
sec14-1), or in recycling between endosomes and the TGN
(tlg2
and vps35
). Growth was examined at
various temperatures by comparing double mutants with their parent
strains (Fig. 7A).

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|
Fig. 7.
Genetic interactions between
ypt6-2 and other mutations. A,
ypt6-2 (ZLY2) was combined with selected trafficking mutants
by crossing ZLY2 with the respective strains (see Table I). Cells were
grown at 25, 30, and 35 °C by spotting 10-fold diluted
cultures onto YPD plates. ypt32ts represents
ypt32A141D/ypt31 (YLX15).
B, pulse-chase analysis of CPY. WT (MSUC-3D) and mutants (as
indicated) were grown at 25 °C to mid-log phase, preincubated at
30 °C for 30 min, and then subjected to pulse-chase analysis at
30 °C as described in the legend to Fig. 2B.
|
|
Synthetic lethality, the phenomenon describing the inviability of
double mutants that are viable with only one of the mutations, was
observed when ypt6-2 was combined with either ypt1
A136D, yip1-2, or
ret2-1. Synthetic lethality usually indicates that the gene
products concerned act in the same functional pathway. Ypt1-GTPase and
Yip1p, a primarily Golgi-localized membrane protein binding Ypt1p and
Ypt31p (40), are involved in docking/fusion of ER-derived vesicles with
an early Golgi compartment, whereas Ret2p, the
-subunit of COPI,
principally acts in ER retrieval (33). Therefore, the results of the
synthetic lethality screen suggested that Ypt6p might function,
directly or indirectly, in anterograde and retrograde transport between
the ER and the Golgi. The same conclusion could be drawn from other
synthetic negative interactions shown in Fig. 7A. In
particular, severe growth inhibition resulted from combining
ypt6-2 with conditional alleles of either SED5 or
USO1 that encode the Golgi syntaxin and a vesicle tethering factor, respectively, for ER-to-Golgi forward transport. Synthetic negative effects on cell growth was also observed by a combination of
ypt6-2 with temperature-sensitive alleles of either
SEC27 or SEC21, the genes for the COPI components
' and
, respectively. Some of the mutant combinations were also
tested for the maturation and sorting of newly synthesized CPY. We
chose to combine ypt6-2 with either
sly1ts, ret1-1, or sec35-1,
mutants whose primary defects at nonpermissive temperature are either
in ER-to-Golgi transport (41), in retrograde transport between Golgi
compartments and the ER (42), or in more than one transport step (43),
respectively. As can be seen in Fig. 7B, maturation and
sorting of CPY was completely unaffected in sly1ts,
ret1-1, and sec35-1 mutants at a permissive
temperature of 30 °C, but combining either of these mutant alleles
with ypt6-2 resulted in an aggravation of the Golgi
trafficking defect and the mis-sorting of CPY seen in the
ypt6-2 mutant alone. The strongest synthetic defects were
seen in the ypt6-2/sec35-1 double mutant. In
addition, high expression of SEC35 from a multicopy vector
was found to partially rescue ypt6-2 mutant cells from
growth inhibition at 35 °C (data not shown), supporting the view
that Sec35p and Ypt6p might act in the same pathway(s).
Severe synthetic growth defects were also observed when the
ypt6-2 allele was combined with either the deletion of
VPS35 or of TLG2 (Fig. 7A). Although
both deletions by themselves, or ypt6-2 alone, allowed cell
growth at 25, 30, and 35 °C, the double mutants did not grow at
35 °C and were completely (ypt6-2/vps35
) or
severely (ypt6-2/tlg2
) growth-inhibited at
30 °C. As the syntaxin Tlg2p and the Vps35 protein are components of
the machinery of endosome-to-Golgi trafficking, these results are in
line with the conclusions drawn from previous studies (7-10, 13)
indicating that Ypt6p has a function also in recycling between
endosomes and the TGN.
 |
DISCUSSION |
Previous studies on the role of Ypt6p in intracellular protein
transport have led to two quite different models, one suggesting an
involvement of this GTPase in recycling between endosome(s) and the
trans-Golgi (7, 8), and the other postulating a regulatory
role in anterograde transport between early Golgi compartments (11,
12). These models were originally built on the phenotypes that resulted
from studies of ypt6 null mutants or mutants expressing a
C-terminally truncated Ypt6p. Although there is experimental support
for the first of the two models (9, 10), it has been difficult to
establish the primary role for Ypt6p because permanent loss of the
GTPase may result in cells bypassing the requirement for Ypt6p or in
masking certain defects. An instructive example for such a situation is
the loss of clathrin heavy chain function in yeast which results in
mis-sorting of vacuolar proteins immediately after clathrin
inactivation, whereas in cells lacking functional clathrin heavy chain,
vacuolar protein sorting is restored (44). With this in mind,
temperature-sensitive ypt6 mutants were created of which
one, ypt6-2, was growth-inhibited already at 35 °C.
Our findings that at nonpermissive temperatures heat-sensitive
ypt6 mutants accumulate apparently core-glycosylated CPY,
ALP, and Myc epitope-tagged Golgi mannosyltransferases Och1p and Mnn1p, and that synthetic lethality results from combining the
ypt6-2 mutant allele with either the
ypt1A136D or the yip1-2 allele seemed to
support a role of Ypt6p played in anterograde ER-to-Golgi transport.
However, further analysis of transport kinetics of CPY and ALP by
pulse-chase experiments revealed that the accumulated proforms of CPY
and ALP resulted from underglycosylation rather than from a defect in
ER-to-Golgi transport. This was further confirmed by subcellular
fractionation analysis of ypt6-2 mutants that showed Golgi
localization of core-glycosylated vacuolar and Golgi-resident enzymes.
By following the fate of newly synthesized secreted proteins at
nonpermissive temperature, it became evident that a substantial
fraction of them reached the extracellular space, indicating that they
had passed the Golgi.
How might rapid loss of Ypt6p function cause Golgi glycosylation
defects? One possibility could be that Ypt6p is required for proper
functioning, stability, or localization of glycosyltransferases. However, in ypt6-2 mutant cells shifted to nonpermissive
temperatures for up to 2 h, we did not find a significant decrease
in the steady-state level of the Golgi glycosyltransferases Anp1p,
Mnn9p, or Van1p, but we observed underglycosylation of the Golgi
mannosyltransferases Och1p and Mnn1p at steady state already 1 h
after temperature shift. Although there is no evidence for the
N-glycans being essential for the functioning of these
glycosyltransferases, proper localization of the cis-Golgi
mannosyltransferase Och1p has been shown previously to involve a
retrograde transport pathway from late to early Golgi compartments
(38), and Mnn1p, which is apparently localized to two distinct Golgi
compartments, was also proposed to cycle between late and early Golgi
compartments (39, 45). It therefore appears likely that a primary role
of Ypt6p is in retrograde Golgi transport, including the recycling of
Golgi glycosyltransferases which, when disturbed as in conditional
ypt6 mutants, would interfere with their proper localization
and hence result in protein glycosylation defects and a slow down of
anterograde transport. Importantly, glycosylation defects were not
detected in ypt6 deletion mutants at permissive temperature
(9, 11), which would have been expected given the role proposed here
for the GTPase. An explanation for this apparent discrepancy could be
that altered distribution or functional impairment of
glycosyltransferases in permanently Ypt6p-lacking cells is compensated
for by an increase of ER-to-Golgi trafficking activity. Rescue of
ypt6 deletion mutants from growth inhibition at high
temperature through intracellular levels of Ypt1p or the expression of
SLY1-20 (12), which we also observed with the
temperature-sensitive ypt6-2 in the present study, support this assumption. The apparent impairment of ER-to-Golgi traffic that we
have observed in the conditional ypt6 mutant at
nonpermissive temperature, therefore, is likely to be a secondary effect.
In support of an involvement of Ypt6p in retrograde Golgi transport is
our genetic data demonstrating synthetic lethality or severe synthetic
negative growth defects after combining the ypt6-2 mutant
allele with either mutant alleles of various genes encoding subunits of
the COPI complex or with sec35-1. Sec35p is part of a
multicomponent protein complex (43, 46, 47), the primary function of
which appears to be in tethering vesicles in retrograde Golgi
transport. Components of this tethering complex have also been
discussed to act in an endosome-to-Golgi protein retrieval (43, 48).
Interestingly, our study revealed genetic interactions between the
ypt6-2 mutant allele and deletions of either TLG2
or VPS35, nonessential genes whose protein products have
been documented previously to fulfill functions in the recycling of
proteins from endosomes to the Golgi (49-53).
Collectively, the biochemical and genetic data obtained with the
temperature-sensitive ypt6 mutants indicate that the GTPase Ypt6p exerts its presumably regulatory role in vesicular protein transport from endosome(s) to the Golgi as well as in retrograde trafficking between Golgi compartments. Although the involvement of
Ypt6p in several transport steps from endosome(s) to early Golgi
compartments had been postulated from studies undertaken previously
(7-10), it is the generation and analysis of the first conditional
ypt6 mutants that allowed us to observe the rather fast
kinetics of transport disturbances, defects in protein glycosylation by
Golgi mannosyltransferases and in protein sorting in the late Golgi
following Ypt6p inactivation. Further detailed analyses of Ypt6p
function have to await the generation of cell-free systems, allowing
the differentiation between different transport steps, but this will be
a difficult task given the complexity of protein recycling between
compartments of the endocytic pathway and the Golgi complex (54).
Interestingly, after several years of investigating Rab6, the mammalian
counterpart of Ypt6p, it seems certain now that in mammalian cells,
retrograde transport from endosomes to the trans-Golgi
network and from late to early Golgi compartments (and possibly to the
ER) is also controlled by this GTPase although in the form of two
different isoforms (16-18). The Sec22p recycling defect that we have
observed in the present study underlines the importance of Ypt6p in
retrograde Golgi transport and, together with some of the genetic data
obtained, leaves open the possibility that Ypt6p is effective also in
retrograde transport from the Golgi to the endoplasmic reticulum.
 |
ACKNOWLEDGEMENTS |
We thank Hans Dieter Schmitt, Renwang Peng,
and Xiaoping Yang for helpful discussions and for providing yeast
strains and plasmids; Ludwig Lehle (University Regensburg) for
anti-chitinase antibody; Sean Munro (MRC Cambridge) for anti-Anp1p
antibody; Rita Schmitz-Salue for technical assistance; Hans-Peter
Geithe for sequencing; and Ingrid Balshüsemann for secretarial help.
 |
FOOTNOTES |
*
This work was supported by the Max Planck Society and by
grants from the Deutsche Forschungsgemeinschaft, the Fonds der
Chemischen Industrie, and the Human Frontier Science Program (to
D. G.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 49-551-201-1496;
Fax: 49-551-201-1718; E-mail: Dieter.Gallwitz@mpi-bpc.mpg.de.
Published, JBC Papers in Press, October 24, 2002, DOI 10.1074/jbc.M209120200
 |
ABBREVIATIONS |
The abbreviations used are:
ER, endoplasmic
reticulum;
TGN, trans-Golgi network;
ORF, open reading
frame;
PNGase, peptide: N-glycosidase;
COP, coat
protein;
SNARE, soluble NSF
(N-ethylmaleimide-sensitive factor) attachment protein
receptor;
WT, wild type;
ALP, alkaline phosphatase.
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