From the Departments of Molecular, Cellular and Developmental Biology and Biological Chemistry, University of Michigan and the Life Sciences Institute, Ann Arbor, Michigan 48109
Received for publication, October 11, 2002, and in revised form, November 20, 2002
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ABSTRACT |
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Autophagy, pexophagy, and the Cvt pathway are
processes that deliver hydrolytic enzymes and substrates to the yeast
vacuole/lysosome via double-membrane cytosolic vesicles. Whereas these
pathways operate under different nutritional conditions, they all
employ common machinery with only a few specific factors assisting in the choice of the delivery program and the membrane source for the
sequestering vesicle. We found that the YKR020w gene
product is essential for Cvt vesicle formation but not for pexophagy or induction of autophagy. Autophagosomes in the ykr020w Autophagy is a catabolic process conserved among yeast, plants,
and animal cells that permits the cell to eliminate unwanted or
unnecessary proteins and organelles and to recycle the components for
reuse (1, 2). The organellar turnover is exclusively accomplished in
the lysosome/vacuole lumen by a wide range of hydrolases capable of
breaking down all cellular constituents (1, 2). Autophagy plays an
essential role during normal physiological processes such as
starvation, cellular differentiation, cell death, and aging, but also
in preventing some types of cellular dysfunction including cancer
(2).
Studies in the yeast Saccharomyces cerevisiae have led to
the identification of a large number of molecular components that form
the autophagic machinery (1). Interestingly, most of these proteins are
also utilized for the cytoplasm to vacuole targeting (Cvt)1 pathway (3, 4), which
assures the delivery of the resident vacuolar hydrolase aminopeptidase
I (Ape1) (5, 6). The same components are also required for peroxisome
degradation, or pexophagy (7). These various processes operate under
different nutritional conditions, but biochemical and morphological
analyses have shown that in all cases the cargo material (precursor
Ape1 (prApe1), bulk cytoplasm or a specific organelle) is sequestered
by a cytosolic double-membrane vesicle (7-11). The basic mechanism
that leads to the formation of this structure, called an autophagosome,
Cvt vesicle, or pexophagosome, is identical in all three pathways and
it can be divided into five discrete steps: vesicle
induction/nucleation, cargo selection/packaging, vesicle
formation/completion, docking/fusion with the vacuole, and subvacuolar
vesicle breakdown (1, 2). In the case of pexophagy and the Cvt pathway,
the cargo may be specifically targeted to the sequestering membrane
where it starts to be enwrapped by a double lipid bilayer. This process
leads to the creation of the cytosolic double membrane vesicle. The completed vesicle docks with the lysosome/vacuole and successively fuses with it. In this way the inner vesicle is liberated into the
lysosome/vacuole lumen where it is finally consumed by hydrolases.
Cellular signals dictate the selection of the cargo material but also
the size of the forming vesicle (9, 12, 13). The serine/threonine
protein kinase Apg1 and its interacting partner Apg13 are two
components that play a part in all three pathways. These proteins seem
to have a central role in determining the specific cellular response to
nutrient conditions (4, 7, 13-15). Phosphorylation and
dephosphorylation reactions mediate the association of Apg1 and Apg13
(13) creating a modular core complex able to interact with factors such
as Apg17, Cvt9, and Vac8 that are specific only for one or two pathways
(13, 16-18) (Table II).
The rest of the components involved in the biogenesis of autophagosomes
and Cvt vesicles include two conjugation systems that lead to the
covalent linkage of the ubiquitin-like protein Aut7 to a molecule of
phosphatidylethanolamine and the formation of a multimeric complex
composed of Apg12-Apg5 and Apg16 (19). In addition, an
autophagy-specific phosphatidylinositol (PtdIns) 3-kinase complex is
involved in the synthesis of PtdIns(3)P that may serve to recruit
downstream effectors that function in autophagy and the Cvt pathway (1,
20-23).
These shared factors and all the regulatory elements localize to a
punctate perivacuolar organelle, also called the preautophagosomal structure (PAS), that is believed to be the formation site of autophagosomes and Cvt vesicles (24-26). Most of the autophagy (Apg)/Cvt proteins are cytosolic and achieve their correct localization by interaction with other factors or by specific binding to lipids such
as phosphatidylethanolamine or PtdIns(3)P (20, 23, 24, 27). Several
lines of evidence suggest that the source of the sequestering vesicles
for autophagy and the Cvt pathway differ at least in part. For example,
Aut7 is needed for nucleation of Cvt vesicles but not autophagosomes;
Aut7 is needed for expansion of the autophagosomal membrane (12).
Similarly, Apg1 appears to have different roles in these pathways; a
catalytic function is needed for the Cvt pathway, but Apg1 may have a
nonkinase role in inducing autophagy (28). With the exception of the
proteins interacting with the Apg1-Apg13 complex, the rest of the
pathway-specific factors are components of vesicular traffic
machineries (Table II). Autophagy, but not the Cvt pathway, requires
the GTP exchange factors, Sec12 and Sec16, and the two COPII coat
subunits, Sec23 and Sec24 (29). That seems to correlate with studies in
mammalian cells indicating that autophagosomes are derived from the
endoplasmic reticulum (30). However, the tSNARE Tlg2, the vSNARE Tlg1,
and the Sec1 homologue, Vps45, are essential for the formation of Cvt
vesicles but not for autophagosome biogenesis (31). That is also true
for three PtdIns(3)P-binding proteins, the two sorting nexins Cvt13 and
Cvt20 plus the transmembrane protein Etf1 (23, 32).
SNARE-mediated fusion events employ additional proteins called
tethering factors that play a role essential in the specificity and
coordination of those reactions (33, 34). In the present study, we have
identified the fourth subunit of the VFT tethering complex that,
together with Tlg2 and Tlg1, is required for a retrieval transport step
back to the late Golgi that is essential for the recycling of the
vSNARE Snc1, but also for the completion of Cvt vesicles. We also show
that in the absence of those fusion factors, prApe1 and its receptor,
Cvt19, together with Cvt9 are not correctly targeted to the PAS.
Strains and Growth Media--
The S. cerevisiae
knockout strains in the BY4742 background used in this study
(ykr020w
PCR-based integrations of the triple HA tag and the 13 × Myc tag at the 3' end of YKR020w, VPS52,
VPS53, and VPS54 were used to generate strains
expressing fusion proteins under the control of their native promoters.
The templates for integration were pFA6a-3HA-TRP1, pFA6a-13Myc-His3MX6,
and pFA6a-13Myc-TRP1 (35). Normal prApe1 processing and vacuolar
morphology were used to confirm the functionality of all genomic fusions.
Strains were grown in YPD (1% yeast extract, 2% peptone and 2%
glucose) or synthetic minimal medium (SMD; 0.67% yeast nitrogen base
without amino acids, 2% glucose, and auxotrophic amino acids as
needed). Nitrogen starvation was carried out in SD-N medium (0.17%
yeast nitrogen base without amino acids and ammonium sulfate and 2% glucose).
Plasmids--
YKR020w flanked by MfeI
sites was generated by PCR and cloned into the EcoRI site of
the pRS416-CuProtA vector (25) behind sequences expressing two IgG
binding domains of protein A (PA) and the CUP1 promoter, and
before the CYC1 terminator. The new plasmid was called
pCuPAYKR020(416). This construction was also transferred as a
KpnI-SacI fragment into a pRS414 plasmid (36) creating pCuPAYKR020(414). All enzymes for manipulation of
DNA were from New England Biolabs (Beverly, MA). Plasmids expressing PA
(pRS416-CuProtA), GFP-Snc1 (pGS416), GFP-Ape1 (pTS466), CFP-Ape1 (pTS470), Cvt19-CFP (pCVT19CFP(414)), YFP-Aut7 (pRS414EYFP-Aut7), CFP-Aut7 (pRS316ECFP-Aut7), GFP-Cvt9 (pTS495 and pCuGFPCVT9(416)), YFP-Cvt9 (pPS97), and CFP-Cvt9 (pPS98) have been described elsewhere (24, 25, 37, 38).
Protein Extraction and Western Blot--
Cells were inoculated
and grown in YPD overnight to early log phase
(A600 = 0.6). Cells from this preculture were
then either grown again in YPD or nitrogen starved in SD-N medium for
3 h. 1 A600 unit of cells was collected by
centrifugation and proteins were precipitated with 500 µl of ice-cold
10% trichloroacetic acid for 30 min. After spinning the samples for 5 min, pellets were washed once with acetone. Pellets were air dried,
resuspended in 100 µl of MURB buffer (50 mM
Na2HPO4, 25 mM MES, pH 7.0, 1% SDS, 3 M urea, 0.5% 2-mercaptoethanol, 1 mM
NaN3, and 0.05% bromphenol blue), and heated at 75 °C
for 10 min. Aliquots of 10 µl were loaded on 8% SDS-PAGE gels and
after Western blotting, membranes were probed with anti-Ape1 polyclonal
antiserum (6).
Fluorescence Microscopy--
Cells grown to early logarithmic
(log) phase in SMD medium or shifted to SD-N for 3 h, were
prepared for fluorescence and stained with FM 4-64 (Molecular Probes,
Eugene, OR) as described previously (39). Fluorescence signals were
visualized with the use of a Nikon E-800 fluorescent microscope (Mager
Scientific, Dexter, MI). The images were captured with an ORCA II CCD
camera (Hamamatsu, Bridgewater, NJ) with the use of Openlab
software (Improvision, Lexington, MA).
Protein A Affinity Isolation--
Cells were first grown
overnight in SMD medium, then diluted with YPD and grown for an 3 additional hours. 50 A600 units of cells were
harvested, converted to spheroplasts, and kept frozen. Spheroplasts
were resuspended and Dounce homogenized in 2 ml of lysis buffer (40)
containing 2 mM phenylmethylsulfonyl fluoride. Cell
lysates were then centrifuged at 13,000 × g for 15 min
and 1.6 ml of supernatant was incubated for 2 h at 4 °C with 20 µl of prewashed IgG Sepharose beads (Amersham Biosciences).
Beads were then washed twice with lysis buffer (40), once with lysis buffer containing 300 mM KCl, once with lysis buffer
containing 500 mM KCl, then once again with lysis buffer
containing 300 mM KCl, and finally 3 times with the initial
buffer. Finally, beads were heated at 75 °C for 10 min in 50 µl of
MURB buffer. After SDS-PAGE and Western blot, membranes were probed
with anti-HA monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA).
For cross-linking experiments, spheroplasts were prepared as above but
not frozen. Instead, they were resuspended in 200 µl of phosphate
buffer (25 mM potassium phosphate, pH 7.4, 200 mM sorbitol, 20 mM phenylmethylsulfonyl
fluoride, 10× Complete EDTA-free protease inhibitor mixture (Roche
Molecular Biochemicals, Indianapolis, IN)) containing 1.5 mM dithiobis(succinimidyl propionate) (Pierce). Suspensions
were incubated for 30 min at room temperature. To quench the
cross-linker, 200 µl of ice-cold 200 mM Tris-HCl (pH 7.4)
were added and tubes were transferred to 4 °C for 5 min. Finally,
1.6 ml of ice-cold dilution solution (187.5 mM KCl, 6.25 mM MgCl2, 1.25% Triton X-100) was added and
after Dounce homogenization, protein A affinity isolation was performed
as above. After SDS-PAGE and Western blot, membranes were probed with
anti-HA, anti-Tlg1 (41), anti-Pep12 (Molecular Probes), or anti-Sed5
(42) antiserum or antibodies.
Miscellaneous Procedures--
The analyses of protease
sensitivity, Prc1 missorting, cell viability under nitrogen starvation
conditions, pexophagy, Pho8 The ykr020w
A genome-wide approach for the identification of yeast protein
complexes by mass spectrometry indicated a putative interaction between
Ykr020 and Vps52/Sac2 (44). Vps52 together with Vps53 and Vps54 form
the so-called VFT complex, a putative tethering factor required for the
retrieval of proteins from the early endosome to the late Golgi (33,
45). The vps52
A defect in prApe1 maturation under vegetative conditions can be caused
either by a block in the Cvt pathway or by inefficient cleavage of the
prApe1 propeptide by proteases after reaching the vacuole lumen. The
vps52
Most vacuolar protein sorting (vps) mutants display
pleiotropic defects in the sorting of additional vacuolar hydrolases
including Pep4 and Prb1 (49-51). As a result, these strains are
severely compromised for vacuolar processing activity. Accordingly, we could not exclude a defect in prApe1 processing because of reduced proteolytic activity of the vacuole. To explore this possibility, we
took advantage of a green fluorescent protein (GFP)-tagged version of
prApe1 that can be used to monitor defects in the Cvt pathway (38). In
wild type cells expressing GFP-Ape1, fluorescence was localized to the
vacuole lumen either when cells were grown in rich medium or after
nitrogen starvation (Fig. 1C). The vps52
With the previous analysis we could not differentiate between a block
in Cvt vesicle formation or fusion of completed vesicles with the
vacuole. This question was addressed with a protease sensitivity
experiment. Yeast cells were converted to spheroplasts and lysed under
conditions that allow lysis of the plasma membrane while retaining the
integrity of subcellular compartments (52). The lysates were then
treated with proteinase K in the absence or presence of detergent.
During Cvt vesicle formation, prApe1 is not yet enclosed within a
membrane compartment and consequently, in mutants affecting this
process, prApe1 is accessible to exogenous proteases. In strains such
as ccz1 Ykr020, Vps52, Vps53, and Vps54 Are Essential during Starvation but
Are Not Required for Autophagy and Pexophagy--
Most of the
machinery required for the Cvt pathway is also exploited by autophagy
and pexophagy (3, 4, 7). Results shown in Fig. 1, A and
B, indicated that prApe1 processing was normal in the
vps52
The observation that prApe1 import in the vps52
The starvation sensitivity suggests that the vps52
There are two explanations for the reduced autophagic activity in the
vps52
Mutations in structural genes encoding vacuolar hydrolases such as
PEP4 and PRB1 result in a starvation-sensitive
phenotype (8, 58). This finding is in agreement with the requirement for Prb1 (proteinase B) in the breakdown of autophagic bodies, the
single membrane subvacuolar vesicles that contain the cytoplasmic cargo
resulting from autophagy (8). To our knowledge, however, the viability
of vps mutants that are blocked in the biosynthetic delivery
of resident hydrolases to the vacuole has not been examined in
starvation conditions. For this reason, we decided to analyze the
viability under starvation conditions of other deletion strains with a
vps defect. We selected four strains that affect different functions of the late endosome without interfering with normal prApe1
transport.2 The
vps4
When cells are shifted from conditions that necessitate peroxisome
function (e.g. oleic acid) to glucose, excess peroxisomes are degraded (7, 59, 60). Peroxisome degradation requires most of the
machinery that is needed for autophagy (7). The specific degradation of
peroxisomes, pexophagy, induced by glucose adaptation or nitrogen
starvation can be monitored by following the degradation of the
peroxisomal matrix protein Fox3 (7). Growing cells were first shifted
for 12 h to a medium containing glycerol, a suboptimal carbon
source for yeast. Peroxisome proliferation was then induced by
transferring cells for 19 h into a medium with oleic acid as the
sole carbon source. Once shifted to SD-N, the excess peroxisomes were
delivered to the vacuole and degraded in wild type cells (Fig.
2E). Pexophagy is blocked in mutants such as
apg1 Snc1 Recycling Is Blocked in ykr020
As previously reported, in wild type cells most of the fluorescent
staining corresponding to GFP-Snc1 was on the plasma membrane (Fig.
4) (37). A fraction of GFP-Snc1 was also
observed in internal cell structures, probably organelles that Snc1
passes through during its recycling pathway (Fig. 4) (37). In contrast,
in the four mutants analyzed, GFP-Snc1 was no longer found on the cell
surface. Because of the co-localization with FM 4-64, it became evident
that Snc1 was mislocalized to the vacuole (Fig. 4). It should be noted
that in cells where the GFP-Snc1 levels were lower, the staining
pattern was primarily cytosolic punctate dots. These dots may represent
vesicles that cannot fuse with the late Golgi compartment; in the
absence of the VFT complex and Ykr020, Snc1 retrieval to the Golgi
complex is blocked resulting in its delivery to the vacuole via
vesicular intermediates. This cytosolic staining pattern is most
evident in the vps52
Another similarity between ykr020w Ykr020 Is Part of the VFT Complex but It Is Not Required for Its
Stability--
The similar trafficking and vacuole morphology
phenotypes suggested that Ykr020 might be involved in the same
transport step as the VFT complex. In addition, a recent proteomic
analysis indicates that Ykr020 directly interacts with Vps52 (44). We
decided to examine if Ykr020 was part of the VFT complex. To do so, we
fused PA with the N terminus of Ykr020. The functionality of this
fusion was demonstrated by complementation of the vacuole fragmentation phenotype of ykr020w
Having established that Vps51 is part of the VFT complex, we decided to
examine the stoichiometry of this protein in the complex. We repeated
the PA-Vps51 affinity isolation experiment using a strain carrying a
copy of the VPS51 gene tagged on its C terminus with the Myc
epitope. PA-Vps51 was unable to pull down Myc-tagged Vps51 indicating
that there is only one Vps51 subunit per VFT complex (Fig.
5B).
Vps52, Vps53, and Vps54 depend on each other for stability. That is, in
the absence of one of those proteins, the other two are rapidly
degraded (46). During our experiments, we noticed that several
phenotypes associated with the loss of the VFT complex were less
prominent in the vps51 There Is a Single VFT Complex Interacting with Tlg1--
It has
been shown that the VFT complex transitionally binds the vSNARE Tlg1
during the docking of early endosome-derived vesicles with the late
Golgi (45). At present, we could not exclude the presence of two VFT
complexes, only one of which contained Vps51. We decided to examine the
interaction of the VFT complex containing Vps51 with Tlg1 to determine
whether it was functioning similar to the previously analyzed VFT
complex (45). Spheroplasts obtained from the strain expressing either
PA-Vps51 or Vps52-HA were incubated for 30 min at room temperature in
the presence of the amine-reactive cross-linker dithiobis(succinimidyl
propionate) prior to lysis. Protein A affinity isolation was then
performed and the presence of Tlg1 was examined by immunoblot. Pep12
and Sed5, the tSNAREs of the late endosome and the cis-Golgi
complex, respectively (37, 42, 66, 67), were used as controls for
nonspecific cross-linking. As can be seen in Fig.
6A, Tlg1 bound the
Vps51-containing VFT complex under conditions where the other tSNAREs
were not cross-linked.
Pulse-chase radiolabeling followed by Ape1 immunoprecipitation has
shown that under rich growth conditions, Tlg2, Tlg1, and the Sec1
homologue, Vps45, are essential for the formation of Cvt vesicles but
not for autophagosome biogenesis (31). This type of experiment
demonstrates that those proteins have a direct impact on the vesicle
formation process. To show that the steady state conditions used for
our investigations were genuinely representative of a direct role of
the VFT complex in Cvt vesicle biogenesis, we decided to examine the
state of Ape1 in tlg2 Cvt9 Is Mislocalized and the Ape1-Cvt19 Complex Is No Longer
Correctly Transported to the PAS in the Absence of the VFT
Proteins--
Most of the Cvt and autophagy pathway components are
required for the vesicle formation/completion step and localize to a perivacuolar punctate structure (24, 25). Cells lacking the VFT complex
are defective in Cvt vesicle biogenesis (Fig. 1D) indicating
that probably one or more proteins necessary for this process are
missing. To explore this possibility, we checked if the localization
pattern of GFP-tagged Apg/Cvt components was altered in
vps51
After synthesis, prApe1 forms a large cytosolic oligomer that binds
Cvt19 (69, 70). This association triggers the transport of this complex
to the site of Cvt vesicle formation in a process that requires Cvt9
(38). Our examination of GFP-tagged Apg/Cvt proteins in
vps51
Because Cvt9 mediates the correct targeting of the prApe1-Cvt19 complex
to the site of Cvt vesicle formation (38), we decided to investigate
the localization of this protein relative to Cvt19 and the PAS. Wild
type and vps52 Vps51 Is a Subunit of the VFT Complex--
The Vps fifty-three
(VFT) complex and the rab GTPase Ypt6 are required for the tethering of
early endosome-derived vesicles with late Golgi membranes (Fig.
8) (33, 45). Ric1 and Rgp1 form another
complex that plays a crucial role during this recognition event. These
two proteins localize to the late Golgi compartment where they recruit
and catalyze nucleotide exchange on Ypt6 (40). Ypt6:GTP then binds
directly to the VFT complex on the incoming vesicles (40). Because the
VFT complex is associated with the vSNARE Tlg1, this tethering
association brings Tlg1 in proximity with Tlg2, the tSNARE on the late
Golgi membranes, leading to the assembly of the SNARE bundle, the core
of the membrane fusion reaction (45).
The VFT complex is composed of three subunits: Vps52, Vps53, and Vps54
(40, 46). In a screen designed to find new deletion strains affecting
the Cvt pathway, we identified Ykr020 (originally named Cvt22) as the
fourth component of this complex and we named it Vps51.
Vps51
We obtained direct evidence that Vps51 is part of the VFT complex
through an affinity isolation analysis. Vps51 tagged with protein A
pulled down each of the other components of the VFT complex (Fig.
5A). Previous work has established that the ratio between
each subunit is 1:1:1 (46). Vps51 did not pull down itself indicating
that there is only one Vps51 subunit per complex (Fig. 5B).
Thus, we concluded that the VFT complex is a heterotetramer with a
1:1:1:1 stoichiometry.
Vps51 is a small 164-amino acid protein that contains 2-3 putative
coiled-coil regions. This protein has characteristics that differ from
those of the other VFT complex components. For example, Vps52, Vps53,
and Vps54 depend on each other to avoid rapid degradation, whereas
Vps51 is not necessary for their stability and conversely, these three
VFT components are not required for maintaining the appropriate
cellular levels of Vps51 (46) (Fig. 5, C and D). The Vps52-Vps53-Vps54 trimer is still present in the absence of Vps51,
which probably accounts for a residual activity of the VFT complex that
would explain the less severe phenotypes observed in the
vps51 The VFT Complex Is Specifically Required for Cvt Vesicle Biogenesis
but Not for Autophagy and Pexophagy--
Autophagy, pexophagy, and the
Cvt pathway utilize a common machinery and only a few factors specific
for one route are known (1, 3, 4, 7) (Table
II). In this article we demonstrate that
the VFT complex is one of those. The Cvt pathway operates during
vegetative growth conditions and it assures the delivery of prApe1 from
the cytosol to the vacuole lumen (5, 6). Under the same conditions, the
absence of the VFT complex causes a block in prApe1 transport and in
its consequent processing (Fig. 1, A and C).
Interestingly, the same cells are able to correctly carry out autophagy
and pexophagy (Figs. 1A and 2, A, C,
and E). There are some delays in the kinetic rates of those
processes with vps51
Tlg2, Tlg1, and Vps45 are also essential for the formation of Cvt
vesicles but not for autophagosome biogenesis (31) (Fig. 6,
B and C). It has been shown that the VFT complex
interacts directly with Tlg1 and indirectly with Tlg2 (45). Here we
demonstrated that indeed, there is a unique VFT complex composed of
four subunits that transiently bind Tlg1 (Fig. 6A). Taking
these results together, we can conclude that the VFT complex, Tlg2, and
by extension Tlg1 and Vps45, are mediating a transport step essential
for the formation/completion of Cvt vesicles.
The absence of the VFT complex results in a block of prApe1 import into
the vacuole under vegetative conditions (Fig. 1C). This
defect is because of an incomplete Cvt vesicle because prApe1 is
accessible to exogenous proteinase K (Fig. 5D). The arrest of the vesicle formation process can be caused either by the lack of
one or more components necessary for Cvt vesicle biogenesis or by the
failure to deliver membranes that are needed for the formation process.
We find that Aut7 is correctly lipidated and localized to the PAS in
vps51
Cvt9 is a specific factor required for the Cvt pathway and pexophagy
but not for autophagy (16) (Table II). Because Cvt9 is mislocalized in
VFT complex mutants, one would expect that pexophagy would be impaired
in the same cells. An interesting observation is that nitrogen
deprivation triggers the correct targeting of Cvt9 to the PAS in the
VFT mutants, furnishing an explanation as to why the prApe1 block is
rapidly bypassed during autophagy (Fig. 7B). As mentioned,
Cvt9 itself is not required for autophagy, but its proper
re-localization may reflect the correct positioning of an associated
factor that is essential for this pathway (16) (Table II). Nitrogen
starvation conditions are also employed to induce peroxisome
degradation (see "Experimental Procedures"). Thus, under those
circumstances, Cvt9 is probably correctly localized so that this
catabolic process proceeds normally (Fig. 2E). That leads us
to conclude that prApe1-Cvt19 and peroxisomes need a common element,
Cvt9, to be targeted to the PAS, but those two pathways have different
trafficking requirements with pexophagy probably using the same
membrane source as autophagy.
The great specificity in vesicular traffic is achieved by the
partnership between SNAREs and tethering factors (33, 34). These
proteins can be involved in one or several different fusion events, but
the combination between them creates a specific assortment utilized
only for a unique vesicular trafficking step. For this reason, Tlg2,
Tlg1, and the VFT complex are probably participating in one or more
retrieval steps back to the Golgi (Fig. 8). Two hypotheses can explain
the role of this fusion machinery in Cvt vesicle assembly. The first
possibility is that there is a retrograde transport route from the PAS
for specific proteins. One possible cargo molecule of this recycling
pathway is Apg9. This transmembrane protein is localized to the PAS but
it is not found on complete autophagosomes, indicating that it is
retrieved prior to the fusion of those vesicles with the vacuole (24,
25, 77). The second hypothesis is that in the absence of this fusion
machinery, the proper homeostasis of either the early endosome or Golgi
complex is severely compromised, and that has an indirect effect on Cvt vesicle completion by altering the sorting of specific transmembrane proteins. Tlg2 also localizes to Cvt vesicles (31). For this reason, at
the moment we cannot exclude that this tSNARE is playing additional
roles in the homotypic fusion that leads to Cvt vesicle completion or
in the transport of intermediate vesicles to the PAS that are required
for Cvt vesicle formation.
Future work will help to connect together all the specific proteins
required for Cvt vesicle assembly and will help to elucidate in better
detail the mechanism that leads to the formation of the PAS. Autophagy,
however, employs factors not required for the Cvt pathway to supply the
PAS with membranes suggesting that there are fundamental differences
between the mechanism of formation of Cvt vesicles and
autophagosomes. The comparison between the trafficking requirements for
PAS formation during vegetative growth with those during starvation
will improve our understanding of the reorganization of these
trafficking routes in different environmental situations.
mutant, however, have a reduced size. We demonstrate that Ykr020 is a subunit of the Vps fifty-three tethering complex, composed of Vps52,
Vps53, and Vps54, which is required for retrograde traffic from the
early endosome back to the late Golgi, and for this reason we named it
Vps51. This complex participates in a fusion event together with Tlg1
and Tlg2, two SNAREs also shown to be necessary for Cvt vesicle
assembly. In particular, those factors are essential to correctly
target the prApe1-Cvt19-Cvt9 complex to the preautophagosomal structure, the site of Cvt vesicle formation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, vps52
, vps54
,
cvt9
, apg9
, vac8
, apg1
, vps4
, vps5
,
vps27
, vps29
, tlg2
and
ccz1
) were from ResGenTM (Invitrogen,
Carlsbad, CA). A vps53
homozygous diploid strain obtained
from the same company was sporulated and dissected to obtain the
VPS53 deletion in a similar background. The rest of the
employed strains are listed in Table I.
For YKR020w and VPS52 gene disruptions, the
entire coding regions were replaced with either the URA3
gene from Kluyveromyces lactis flanked by coliphage loxP
sites or the HIS5 gene of Schizosaccharomyces
pombe, using PCR primers containing ~40 bases of identity to the
regions flanking the open reading frame. The
vac8
::URA3 disruption cassette,
generously provided by Dr. Lois Weisman (University of Iowa), was
digested with AflII and EcoRI and the reaction
mixture was then used to transform the vps52
strain.
Double deletants were selected by verifying the absence of Vac8 in the
cell extracts with anti-Vac8 immunoblots (18).
Saccharomyces cerevisiae strains used in this study
60 activity, and electron microscopy
using Spurr's resin (Ted Pella, Redding, CA) for embedding were
conducted as described previously (7, 23, 27, 43).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, vps52
, vps53
, and vps54
Mutants Block
Precursor Ape1 Maturation Only Under Rich Growth Conditions--
We
identified the YKR020w gene in a genomic approach where
nonessential gene deletions in the yeast S. cerevisiae were
scored for a defect in precursor Ape1 (prApe1) maturation. The
ykr020w
cells grown in rich medium accumulate prApe1,
indicating a defect in the Cvt pathway (Fig.
1A). Certain apg
and cvt mutants that are defective in prApe1 transport under
vegetative conditions are able to import the protein into the vacuole
when autophagy is induced by starvation. For example, mutants specific
for the Cvt pathway such as cvt9, vac8,
cvt13, and cvt20 or mutants that block
autophagosome expansion but not induction such as aut7 are able to mature prApe1 when shifted to starvation conditions (12, 16,
17, 23). We examined processing of prApe1 in the ykr020w
mutant following induction of autophagy. The ykr020w
strain displayed normal processing of prApe1 when this strain was
shifted to a nitrogen starvation medium (Fig. 1A),
indicating that autophagic induction is not impaired.
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Fig. 1.
The ykr020w ,
vps52
, vps53
, and
vps54
cells have a defect in Cvt vesicle
completion. A, The ykr020w
,
vps52
, vps53
, and vps54
strains have a reversible inhibition of prApe1 processing similar to
that of vac8
cells. Wild type (WT),
ykr020w
, vps52
, vps53
(FRY107), vps54
, apg9
, and
vac8
cells in the BY4742 background grown either in YPD
or nitrogen starved in SD-N medium for 4 h were trichloroacetic
acid precipitated. Acetone-washed proteins were then resolved by
SDS-PAGE and prApe1 maturation analyzed by immunoblot. B,
Prc1 is mislocalized to the periplasmic space in the
ykr020w
mutant similarly to vps52
cells.
Cells from wild type, ykr020w
, and vps52
strains in the BY4742 background were pulse-labeled for 10 min and
chased for 30 min. Internal (I) and external (E)
Prc1 was immunoprecipitated and then resolved by SDS-PAGE.
C, The ykr020w
, vps52
,
vps53
, and vps54
strains have a reversible
accumulation of GFP-prApe1 in a cytosolic punctate structure. WT,
ykr020w
, vps52
, vps53
(FRY107), and vps54
strains in the BY4742 background were
transformed with a plasmid expressing the N-terminal GFP-tagged prApe1
(pTS466). Transformed cells were grown either in SMD or nitrogen
starved for 3 h in SD-N medium and examined with a fluorescence
microscope. DIC, differential interference contrast.
D, precursor Ape1 is protease-sensitive in the
vps52
mutant. The vps52
, vps54
,
ykr020w
, and ccz1
strains in the BY4742
background were grown in YPD until early log phase. The cells were
converted to spheroplasts and lysed. The total cells lysates
(T) were centrifuged at 13,000 rpm for 5 min to separate the
pellet (P13) and the supernatant (S13) fractions.
The P13 fraction was resuspended in lysis buffer (27) and then mixed
with equal volumes of lysis buffer, 40 µg/ml proteinase K, 0.4%
Triton X-100, or proteinase K plus 0.4% Triton X-100, and incubated on
ice for 30 min. The reactions were stopped by adding trichloroacetic
acid. Samples were resolved by SDS-PAGE and examined by immunoblot with
serum to Ape1. The ccz1
control cells accumulate
completed Cvt vesicles and prApe1 is accessible to proteinase K only in
the presence of Triton X-100. Even in the absence of detergent, prApe1
in the vps52
strain was digested to its mature form by
the same protease indicating that Cvt vesicles were not completely
assembled. Essentially identical results were obtained for the
vps52
and ykr020w
strains. The T,
S13, and P13 fractions were also probed for the
cytosolic marker Pgk1. Recovery of Pgk1 in the S13 fractions indicates
efficient lysis of spheroplasts.
and vps54
mutants were also
identified in our screen that detected the prApe1 defect in the
ykr020w
strain. Furthermore, the vps52
and
vps54
cells displayed a similar property in reverting the
prApe1 maturation defect after nitrogen starvation (Fig.
1A). In contrast, vps53
cells showed normal
prApe1 processing in all media. However, further analysis of the
commercial vps53
haploid strain supplied by ResGen indicated that the deleted gene was not VPS53. To analyze
the prApe1 phenotype in the vps53
strain, we sporulated
the ResGen vps53
homozygous diploid strain. The resulting
haploid spores showed the same reversible block in prApe1 maturation
that was observed for the ykr020w
, vps52
,
and vps54
strains (Fig. 1A).
, vps53
, and vps54
mutants missort carboxypeptidase Y (Prc1) (46, 47). This missorting is
caused by a block in retrieval of Vps10 to the Golgi complex (46); Vps10 is the receptor required for Prc1 transport to the vacuole (48).
We examined the sorting of Prc1 in the ykr020w
mutant to
determine whether it displayed a similar phenotype. Cells from the
ykr020w
strain were converted to spheroplasts, subjected to pulse-chase labeling, and immunoprecipitated with antiserum to Prc1.
In wild type cells, Prc1 was correctly delivered to the vacuole and
processed to the mature form; essentially no missorting was observed
(Fig. 1B). In agreement with the published data, vps52
cells secreted part of Prc1 as the p2
(Golgi-modified) form (Fig. 1B) (46). The result obtained
with ykr020w
cells was very similar to that seen with the
vps52
strain (Fig. 1B).
, vps53
, vps54
, and ykr020w
strains have fragmented vacuoles (46, 47) (Fig. 4). Nonetheless, in
contrast to the wild type strain, we could determine that when
vps52
, vps53
, vps54
, and ykr020w
cells expressing the same GFP-Ape1 chimera were
grown in rich medium, GFP-Ape1 was concentrated at a perivacuolar
punctate structure (Fig. 1C). This site may correspond to
the PAS (24, 25). The PAS is generally enhanced in mutants with a
defect either in the formation of Cvt vesicles or in their fusion with the vacuole (24, 25). This result indicates that the missorting of
vacuolar proteases in the vps52
, vps53
,
vps54
, and ykr020w
strains does not account
for the accumulation of prApe1. Rather, this defect is because of a
block of prApe1 import into the vacuole lumen. Transport of GFP-Ape1
into the vacuole lumen could be restored after transferring the same
cells into medium lacking nitrogen (Fig. 1C), in agreement
with the reversibility of the prApe1 processing phenotype.
that have a block in fusion (53, 54), prApe1 is
enwrapped by the membrane of the Cvt vesicle and therefore isolated
from the cytosol. As shown in Fig. 1D, in ccz1
cells prApe1 was protected from proteinase K. In contrast, prApe1 was
fully accessible to the proteolytic action of the same proteinase in
the vps52
strain. Identical results were also obtained with vps54
and ykr020w
cells (data not
shown). We concluded that the vps52
, vps54
,
and ykr020w
mutants, and by extension the
vps53
mutant, are blocked at the formation/completion
step of Cvt vesicle biogenesis.
, vps53
, vps54
, and
ykr020w
strains when autophagy was induced by nitrogen
starvation. Recently, Ishihara et al. (29) reported that the
Cvt pathway is operative and autophagy blocked during nitrogen
deprivation in a sec12 mutant. Accordingly, we could not
exclude the possibility that prApe1 maturation in the
vps52
, vps53
, vps54
, and
ykr020w
strains under starvation conditions was because
of the Cvt pathway. To explore this possibility, we took advantage of
vac8
, a mutant that is defective for the Cvt pathway but
not for autophagy (17). We made a vps52
vac8
double mutant to see if prApe1 transport still
occurred when those cells were starved for nitrogen. Precursor Ape1
processing was analyzed by Western blot in wild type,
vps52
, vac8
, and vps52
vac8
cells grown either in the presence or absence of
nitrogen. As expected, in both vps52
and
vac8
cells prApe1 transport was blocked when the strain
was grown in rich medium and restored when cells were shifted to a
medium lacking nitrogen (17) (Fig. 2A). A similar result was
obtained with the vps52
vac8
double mutant
(Fig. 2A). The ability to transport prApe1 to the vacuole under nitrogen starvation conditions in the absence of Vac8 indicated that in vps52
cells this process was performed by
autophagy and not by the Cvt pathway.
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Fig. 2.
The ykr020w ,
vps52
, vps53
, and
vps54
mutants are specifically defective in the Cvt
pathway. A, precursor Ape1 is not imported to the
vacuole by the Cvt pathway under starvation conditions in the
vps52
strain. Wild type (WT),
vps52
, vac8
, and vps52
vac8
(FRY119) cells in the BY4742 background were
analyzed as in Fig. 1A. The vps52
vac8
double mutant was able to import and mature prApe1.
Y, YPD; -N, SD-N. B, the ykr020w
and VFT mutant strains are sensitive to nitrogen starvation. Wild type,
ykr020w
, vps52
, vps53
(FRY107), vps54
, cvt9
, and
apg1
strains in the BY4742 background were grown in YPD
medium until early log phase and then shifted to SD-N. At the indicated
time points, equal volumes of culture were withdrawn and plated on YPD
plates. After 3 days the number of colonies representing the viable
cells was counted, and the percentage of survival calculated and
plotted against time in starvation medium. C, autophagy is
functional in the vps52
and ykr020w
mutants. Wild type (TN124), ykr020w
(FRY122),
vps52
(FRY122), and apg13
(D3Y103) cells
expressing Pho8
60 were shifted from YPD medium (black
bars) to SD-N medium (white bars) for 4 h.
Autophagy induction was determined by a Pho8 activity assay. Results
were expressed as percentage of the activity measured for the wild type
strain starved for nitrogen. Error bars represent the
standard deviation from three separate experiments. D,
viability of vps mutants is sensitive to nitrogen
starvation. Wild type (WT), vps4
,
vps5
, vps27
, and vps29
strains in the BY4742 background were treated as in panel D. E, the ykr020w
, vps52
, and vps53
strains are not
defective in pexophagy. Cells from wild type, apg1
,
ykr020w
, vps52
, and vps53
(FRY107) strains in the BY4742 background were grown in conditions that
induce peroxisomes, washed, and resuspended in SD-N medium for the
times indicated. The presence of the peroxisomal thiolase enzyme, Fox3,
was detected by immunoblotting.
strain is
dependent on autophagy cannot be interpreted as indicating that autophagy is fully functional in this strain; prApe1 can be transported to the vacuole in an autophagy-dependent process even in
situations where autophagy is severely impaired (12, 17). For example, in the absence of Aut7, induction of autophagy leads to the formation of abnormally small autophagosomes that can transport prApe1 to the
vacuole but that cannot maintain a normal level of autophagy (12). We
examined survival of the vps52
, vps53
,
vps54
, and ykr020w
strains under starvation
conditions as another method for assessing the autophagic capacity of
these mutants. Strains that are defective in autophagy display limited
viability under starvation conditions (55). The wild type,
apg1
(15), cvt9
(16), vps52
,
vps53
, vps54
, and ykr020w
strains were grown to mid-log phase, shifted to nitrogen starvation
conditions, and their viability was determined over time. The wild type
strain survived nitrogen starvation for more than 12 days without a
significant decrease in viability (Fig. 2B). The
cvt9
strain, defective primarily in the Cvt pathway, was
also relatively resistant to starvation (16). In contrast, the number
of viable cells decreased dramatically in the VFT complex and
ykr020w deletion strains over the same time period. This
rapid loss in viability was similar to that seen with
apg1
cells, a strain that is extremely sensitive to starvation (55).
,
vps53
, vps54
, and ykr020w
strains may be partially defective in autophagy. Alternatively, this
phenotype could be because of other defects. Under conditions of
nutrient stress it becomes necessary for the cell to transport
cytoplasm to the vacuole by autophagy. However, subsequent to vacuolar
delivery, these components must be degraded to generate an internal
supply of nutrients (8). Cells require a fully functional vacuole to
degrade and transport the recycled material back to the cytosol. The
vps52
, vps53
, vps54
, and ykr020w
mutants affect the proper delivery of resident
vacuolar hydrolases (46, 47) (Fig. 1). For this reason, it could not be
excluded that the loss of viability observed after nitrogen starvation
was caused by the inability to degrade the cytoplasmic substrates that
had been delivered through autophagy. To directly quantify autophagy,
we decided to measure the vacuolar processing of the cytosolic marker
protein Pho8
60. This truncated form of the vacuolar alkaline
phosphatase (Pho8) lacks the transmembrane domain and consequently
localizes to the cytosol (56). This protein is delivered to the vacuole
exclusively by autophagy. Proteolytic cleavage of the Pho8
60
propeptide in the vacuole lumen generates the active form of the
enzyme, which can be detected by an activity assay (56). The
YKR020w and VPS52 genes were knocked out in a
strain where the chromosomal PHO8 gene was replaced with
pho8
60, and phosphatase activity was
determined either before or after nitrogen starvation. In wild type
cells, there was low alkaline phosphatase activity when cells were
grown in rich medium (Fig. 2C). There was an ~7-fold
increase in activity following the induction of autophagy when cells
were shifted to SD-N. Apg13 is essential for autophagy (14, 17). In
contrast to wild type cells, there was no increase in alkaline
phosphatase activity from Pho8
60 when apg13
cells were
shifted to starvation conditions. As shown in Fig. 2C,
autophagy in the ykr020w
and vps52
strains was reduced to 75 and 60%, respectively, of the wild type levels but
was not completely abolished compared with apg13
cells.
This result indicates that autophagy was at least partially active in
the vps52
and ykr020w
mutants.
and ykr020w
strains. First, these
differences may reflect a slower cellular metabolism and as a result,
deletion strains form a reduced number of autophagosomes compared with wild type cells during the same time period. In fact, the
vps52
, vps53
, and vps54
strains grow substantially slower than wild type cells, whereas the
ykr020w
mutant has an intermediate doubling rate (data
not shown). The second possibility is that in the mutant cells,
autophagosomes are smaller and consequently they are able to transport
less material into the vacuole. Accordingly, we decided to analyze the
morphology of the autophagosomes in the mutant strains by electron
microscopy to determine their size and number. Autophagosomes are
transient structures that fuse with the vacuole. To stabilize cytosolic
autophagosomes we took advantage of a conditional allele of
VAM3, a gene coding for a tSNARE required for the fusion of
Cvt vesicles and autophagosomes with the vacuole (57). Cells were grown
in YPD medium at 26 °C to early log phase and then shifted to
37 °C for 3 h either in the same medium or in SD-N medium. All
three strains showed the absence of autophagosomes when grown in rich
medium (Fig. 3). Under nitrogen
starvation conditions, however, vps52
and
ykr020w
cells accumulated smaller autophagosomes than
those present in the wild type strain (Fig. 3). The total number of
autophagosomes in the mutant strains, however, appeared comparable with
that in the wild type strain. We therefore concluded that reduced
autophagic activity in the ykr020w
, vps52
,
vps53
, and vps54
strains is caused by
the inability to assemble normal sized autophagosomes.
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Fig. 3.
The ykr020w and
vps52
strains have smaller autophagosomes.
Wild type (WT, TDY2), ykr020w
(FRY124), and
vps52
(FRY125) cells were grown in YPD medium at 26 °C
to early log phase. Cultures were split in half and centrifuged. One
sample was resuspended again in YPD medium whereas the other was
resuspended in SD-N medium. Cells were then grown at 37 °C for
3 h. Permanganate fixation, dehydration, and embedding were
carried out as described by Kaiser and Schekman (78). Uranyl
acetate-stained sections were observed using a Philips CM10
transmission electron microscope. Examples of autophagosomes are
indicated with an arrow. The bar is 1 µm.
, vps5
, vps27
, and
vps29
cells all lost viability very quickly, similar to
what was observed for vps52
, vps53
, vps54
, and ykr020w
cells (Fig.
2D). This result confirmed our hypothesis that normal
traffic to the vacuole is required to utilize the potential nutrient
pool created by autophagy and provides a likely explanation for the
starvation sensitivity of strains such as vps52
,
vps53
, vps54
, and ykr020w
that do not appear to be defective for autophagy.
where autophagy is defective, and consequently Fox3 levels remain unchanged (7) (Fig. 2E). Fox3 degradation in ykr020w
, vps52
, vps53
, and by
extension vps54
cells was essentially normal with only
some minor differences in the degradation rate relative to wild type
cells (Fig. 2E). We concluded that the VFT complex and
Ykr020 are not required for pexophagy.
, vps52
, vps53
, and
vps54
Cells--
Vps52, Vps53, and Vps54 are subunits of the VFT
complex and in their absence the transport step between the early
endosome and late Golgi is blocked (40). Snc1 is a member of the vSNARE family and it mediates fusion of exocytic vesicles with the plasma membrane (37, 61). Snc1 is mostly at the cell surface, but it undergoes
rapid endocytosis and is transported from the early endosome to the
late Golgi where it is reused (37). Mutants that affect early endosome
function and transport from this compartment mislocalize Snc1 (37, 40,
45, 62, 63). We transformed wild type, vps52
,
vps53
, vps54
, and ykr020w
cells with GFP-Snc1 (37) to see if the cycling of this chimera was
affected. Cells were also stained with FM 4-64, a lipophilic
fluorescent dye that allows visualization of the yeast vacuole
(64).
and vps54
strains
(Fig. 4). Default delivery of Snc1 to the vacuole is similar to the
fate observed for the Vps10 receptor in VFT mutant cells (46).
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Fig. 4.
The ykr020w ,
vps52
, vps53
, and
vps54
cells have a defect in Snc1 recycling and
display abnormal vacuole morphology. Wild type (WT),
ykr020w
, vps52
, vps53
(FRY107), and vps54
cells in the BY4742 background were
transformed with a plasmid expressing the GFP-Snc1 chimera (pGS416).
Transformed cells were grown to a mid-log stage in SMD medium and FM
4-64 was added to the culture medium for 15 min. Cells were then
collected, incubated in the same medium without FM 4-64 for an
additional 30 min to chase the dye, and finally imaged with a
fluorescence microscope. In wild type cells, Snc1 was concentrated on
the plasma membrane while it was redirected to the vacuole lumen in the
mutants. Pictures represent cells with an average level of GFP-Snc1
expression. In cells where those levels were lower, the staining
pattern was primarily cytosolic punctate dots. FM 4-64 staining showed
that deletion strains have a tubular, fragmented morphology of the
vacuole. DIC, differential interference contrast.
cells and VFT complex
mutants is the structure of the vacuole. Wild type cells typically display a single large vacuole or a multilobed organelle depending on
osmotic conditions (65). In contrast, this organelle is highly fragmented in vps52
, vps53
, and
vps54
cells (46, 47). FM 4-64 staining and differential
interference contrast microscopy along with electron microscopy
confirmed the fragmented vacuole morphology of the vps52
,
vps53
, and vps54
strains and showed that
ykr020w
cells have a similar vacuole morphology but with a slightly less severe fragmentation (Figs. 3 and 4). Taken together with the analysis of Prc1 sorting, these data indicate that the ykr020w
cells have essentially identical trafficking
defects as vps52
, vps53
, and
vps54
mutants in regard either to prApe1 transport or
retrieval from the early endosome.
cells (not shown). This construct
was then used to transform three different strains, each carrying one
subunit of the VFT complex tagged at its C terminus with a triple
HA epitope. Spheroplasts obtained from those strains were lysed
and the PA chimera isolated using IgG-Sepharose beads. Bound complexes
were released from the Sepharose by boiling it in sample buffer, and the presence of HA-tagged proteins was tested by immunoblotting. As
shown in Fig. 5A, each one of
the VFT complex subunits was selectively pulled down by the PA-Ykr020
fusion indicating that Ykr020 was part of this complex. During the
writing of this article a review article was published stating that
YKR020w was allelic with VPS51 (33). Accordingly,
we decided to name the YKR020w gene VPS51 to
avoid future confusion.
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Fig. 5.
The YKR020w gene product is
a component of the VFT complex. A, Ykr020 binds Vps52,
Vps53, and Vps54. Strains expressing Vps52-HA (PSY118), Vps53-HA
(PSY119), or Vps54-HA (PSY120) and carrying a plasmid expressing either
the PA-Ykr020 construct (pCuPAYKR020(416)) or PA alone (pRS416-CuProtA)
were used to prepare detergent-solubilized extracts (Ext) as
described under "Experimental Procedures." IgG-Sepharose beads were
used to affinity purify the PA fusions together with the associated
proteins (IP). Eluted polypeptides were separated with
SDS-PAGE and then visualized by immunoblotting with antiserum to HA.
For each experiment 0.2% of the total lysate or 20% of the total
eluate were loaded per gel lane. B, there is only one Ykr020
molecule per VFT complex. The experiment performed in panel
A was repeated with a strain (PSY116) expressing Ykr020-Myc.
C, Ykr020 is not required for the stability of Vps53. Wild
type (WT, PSY119), vps52 (FRY116), and
ykr020w
(FRY117) strains expressing Vps53-HA were grown
to early log phase and proteins were precipitated with trichloroacetic
acid. Proteins were separated by SDS-PAGE and analyzed by Western blot
with anti-HA antibodies. Pgk1 was used to verify that the same amount
of material was loaded on each gel lane. D, Vps52 is not
necessary to maintain normal cellular levels of Ykr020. Wild type
(WT, PSY116) and vps52
(FRY118) cells
expressing Ykr020-Myc were analyzed as in panel C using
anti-Myc antibodies.
strain. For example, the
vps51
cells grew better, had less fragmented vacuoles
(Fig. 4), survived longer in media lacking nitrogen (Fig.
2A), and displayed more rapid pexophagy (Fig.
2C). Accordingly, it seemed possible that Vps51 was
dispensable for the stability of the other subunits; in the absence of
Vps51, the VFT complex was still able to have a residual activity. To
investigate this hypothesis, we disrupted either VPS51 or
VPS52 in the strain carrying HA-tagged Vps53. As shown in
Fig. 5C, Vps53 levels were dramatically decreased in
vps52
cells whereas they remained unchanged in the
vps51
strain. We also decided to analyze the stability of
Vps51 in cells lacking one of the three other components. To carry out
this analysis we deleted the VPS52 gene in the strain
expressing Myc-tagged Vps51. In the absence of Vps52, Vps51 levels were
unchanged compared with wild type cells (Fig. 5D). We
concluded that Vps51 is not necessary for the stability of the other
components of the VFT complex and conversely, the other VFT components
are not required for maintaining the appropriate cellular levels of Vps51.
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Fig. 6.
The Vps51-containg VFT complex interacts with
Tlg1. A, Tlg1 interacts with the Vps51-containing VFT
complex. Spheroplasts from wild type (PSY118) cells expressing Vps52-HA
and transformed with either the plasmid expressing PA-Vps51
(pCuPAYKR020(416)) or PA alone (pRS416-CuProtA) were treated with 1.5 mM of the cross-linker (dithiobis(succinimidyl propionate)
(DSP), detergent solubilized, and the PA fusions were
affinity purified on IgG-Sepharose as described in the legend to Fig.
5A. Samples from the extracts (Ext) or purified
fractions (IP) were analyzed by Western blot using anti-HA,
Tlg1, Sed5, and Pep12 antibodies or antiserum. A longer film exposure
is also shown to demonstrate the total absence of cross-linking between
the VFT complex and the control tSNAREs. B, the
tlg2 strain accumulates prApe1 in YPD medium and this
defect is bypassed in nitrogen starvation conditions. Wild type (WT)
and tlg2
cells in the BY4742 background were essentially
treated as in Fig. 1A. C, the tlg2
strain has a reversible accumulation of GFP-prApe1 in a cytosolic
punctate structure. The same strains used in panel B were
transformed with the plasmid carrying the GFP-prApe1 construct (pTS466)
and analyzed as described in the legend to Fig. 1C.
DIC, differential interference contrast.
cells. As expected, prApe1
processing was blocked when tlg2
cells were grown in rich medium and was restored after nitrogen starvation (Fig. 6B).
This result was confirmed by analyzing the GFP-Ape1 chimera in the same
cells under the same growth conditions (Fig. 6C). These
results are identical to those that we obtained with the VFT complex
components (Fig. 1, A and C). Because Tlg1 and
Tlg2 also interact with the VFT complex (45) (Fig. 6A), it
is reasonable to assume that all of these proteins are involved in the
same fusion event essential for Cvt vesicle biogenesis.
and vps52
cells. Of the different
proteins analyzed, only Cvt9, a protein specific for the Cvt pathway
and pexophagy (16), showed a different cellular distribution. Wild type
cells transformed with a plasmid expressing a GFP-Cvt9 chimera under
the control of the strong CUP1 promoter (68) showed GFP fluorescence at a perivacuolar punctate structure (Fig.
7A). In contrast,
vps51
cells carrying the same construct displayed the presence of several dispersed fluorescent dots (Fig. 7A).
This pattern was because of the absence of Vps51 because when the same cells were transformed with a plasmid expressing the PA-Vps51 fusion,
the correct Cvt9 localization was restored (Fig. 7A). This
phenotype was not caused by the overexpression of the GFP-Cvt9 chimera
by the CUP1 promoter because the same fusion under the control of the endogeneous CVT9 promoter gave identical
results although the fluorescent signal was fainter (Fig.
7A). After shifting cells for 3 h to a medium lacking
nitrogen, GFP-Cvt9 became a single fluorescent spot in the
vps51
strain (Fig. 7B). Thus, starvation
conditions that bypass the prApe1 block in VFT complex mutants were
able to induce the correct targeting of Cvt9. The GFP-Cvt9 chimera
displayed similar patterns in vps52
, vps53
, vps54
, and tlg2
strains (data not
shown).
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Fig. 7.
In the absence of the VFT complex, the
prApe1-Cvt19-Cvt9 complex is not properly targeted to the PAS.
A, Cvt9 is mislocalized in the absence of Vps51. Wild type
(WT, SEY6210) and vps51 (FRY126) cells were
co-transformed with the following pairs of plasmids: pRS414 (empty
vector) and pTS495 (promGFP-CVT9); pCuPAYKR020 (414) (pPA-VPS51) and
pTS495; pRS414 and pCuGFPCVT9(416) (pCuGFP-CVT9); pCuPAYKR020(414) and
pCuGFPCVT9(416). The transformed cells were grown in SMD medium to
early log phase and visualized with a fluorescence microscope. It
should be noted that there are additional very faint dots in the
vps51
cells expressing GFP-Cvt9 under the control of its
native promoter (promGFP-CVT9) that are easily detected when the strong
CUP1 promoter drives expression of the same chimera.
B, Cvt9 mislocalization in vps51
cells is
reversed by nitrogen starvation conditions. The vps51
strain in the BY4742 background was transformed with pCuGFPCVT9(416)
(pCuGFP-CVT9). Transformed mutants were either grown in SMD medium to
early log phase or starved for nitrogen in SD-N medium for 3 h and
then analyzed with a fluorescence microscope. Identical results were
obtained with tlg2
, vps52
,
vps53
, and vps54
strains in the BY4742
background (data not shown). C, the prApe1-Cvt19 complex is
not correctly targeted to the PAS in the absence of the VFT complex.
Wild type (SEY6210) and vps52
(PSY113) strains were
transformed with the following two pairs of plasmid: pTS470 (CFP-Ape1)
and pRS414EYFP-Aut7 (YFP-Aut7); pCVT19CFP(414) (Cvt19-CFP) and
pRS414EYFP-Aut7. Transformed cells were either grown in SMD medium to
early log phase or starved for nitrogen in SD-N medium for 3 h and
then visualized with a fluorescence microscope. The CFP-Ape1 and
Cvt19-CFP did not co-localize with the PAS (YFP-Aut7) when cells were
grown in SMD medium. Nitrogen starvation conditions induced the correct
targeting of CFP-Ape1 and Cvt19-CFP to the PAS because those two
chimeras were in the same punctate structure as YFP-Aut7 in all cells.
These observations were confirmed by repeating the same experiment in
vps51
and vps53
cells (data not shown).
D, Cvt9 is associated with the prApe1-Cvt19 complex away
from the PAS in the vps52
mutant. Wild type (SEY6210) and
vps52
(PSY113) strains were transformed with the
following two pairs of plasmid: pPS98 (CFP-Cvt9) and pRS414EYFP-Aut7
(YFP-Aut7); pPS97 (YFP-Cvt9) and pCVT19CFP(414) (Cvt19-CFP).
Transformed cells were grown in SMD medium to early log stage and
analyzed with a fluorescence microscope. The CFP-Cvt9 fluorescent dots
were separated from the PAS (YFP-Aut7), whereas co-localization between
Cvt19 and Cvt9 was observed in all the images taken. DIC,
differential interference contrast.
and vps52
cells indicated that prApe1
and Cvt19 were restricted to a single punctate structure (Fig.
1C and data not shown). However, this observation could not
rule out the possibility that the fluorescent structure observed with GFP-Ape1 or GFP-Cvt19 was not correctly targeted to the site of Cvt
vesicle formation, especially considering the observation that Cvt9 is
mislocalized in vps51
and VFT mutant cells. For this
reason we decided to transform the vps52
strain with
YFP-Aut7 and either Cvt19-CFP or CFP-Ape1. Analysis of Aut7 provides an independent way to mark the site of vesicle formation (24, 71, 72). As
predicted, in wild type cells grown in rich medium Cvt19 and prApe1
were both in the same structure as Aut7 (Fig. 7C) (25, 38),
whereas in the vps52
strain those two proteins no longer co-localized with Aut7 in 40-50% of the cells examined (Fig.
7C). Nitrogen starvation conditions reversed the defect and
resulted in localization of the prApe1-Cvt19 complex to the correct
destination (Fig. 7C).
cells were transformed either with
Cvt19-CFP and YFP-Cvt9 or with YFP-Aut7 and CFP-Cvt9. As expected, in
wild type cells grown either in rich medium or starved for nitrogen,
Cvt19 and Aut7 were both in the same location as Cvt9, e.g.
the PAS (Fig. 7D) (25, 38). In contrast, in the absence of
VPS52, Cvt9 maintained the co-localization with Cvt19 but
not with Aut7 in 30% of the cell population (Fig. 7D).
These observations suggest that at least one of the causes of the Cvt pathway block in vps52
cells is the inability to
correctly target the prApe1-Cvt19-Cvt9 complex to the site of vesicle
formation. Nitrogen deprivation was once again able to reverse the
defect and direct the prApe1-Cvt19-Cvt9 complex to the Aut7-containing structure (data not shown).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (24K):
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Fig. 8.
Model for VFT complex function in the Cvt
pathway. The VFT complex, including Vps51, functions as a
tethering factor that is required for retrieval (retrograde traffic)
from the early endosome to the Golgi complex (33, 45, 46). In the Cvt
pathway, mutations in the VFT proteins result in mislocalization of the
PAS component Cvt9, suggesting a tethering role in anterograde
transport from the Golgi complex to the PAS. By analogy with its role
in retrieval from the endosome, it is also possible that the VFT
complex is needed for retrograde recovery of certain components from
the PAS. Most of the Apg/Cvt proteins that localize to the PAS are not
found in completed Cvt vesicles suggesting that they are excluded from
the vesicle and retained at the PAS or that they are recycled back to
another compartment such as the Golgi complex. See text for
details.
, but also vps52
, vps53
,
and vps54
, cells have a defect in prApe1 maturation when
grown in rich medium (Fig. 1A). We started to consider the
possibility that Vps51 was interacting with the VFT complex when the
results of a genome-wide approach for the identification of yeast
protein complexes by mass spectrometry appeared indicating a putative
interaction between Vps51 and Vps52 (44). We performed three different
analyses that confirmed that Vps51 was participating in the same
sorting step as the VFT complex. Vps51
cells missort a
population of Prc1 to the periplasmic space, have a fragmented vacuole,
and fail to correctly recycle the vSNARE Snc1, phenotypes that are
shared by the vps52
, vps53
, and
vps54
strains (46, 47) (Figs. 1A and 4). The
Prc1 and vacuolar morphology results are also corroborated by two
different genome-wide studies where all nonessential genes necessary
for Prc1 sorting and homotypic vacuole fusion were identified (65, 73).
Snc1 needs to be retrieved from the early endosome back to the late
Golgi to be reused for exocytosis (37). Tethering/fusion partners of
the VFT complex, Tlg1, Tlg2, Ypt6, Ric1, and Rgp1, are essential for this transport route (37, 40). In the present study we show that the
VFT complex and Vps51 are also required for this recycling step (Fig.
4).
strain (Figs. 2, A and C, and
4). Another peculiarity is that Vps52, Vps53, and Vps54 have clear
mammalian homologues whereas Vps51 does not (33, 74). In a paper
submitted in parallel with ours, Siniossoglou and Pelham (73) show that
Vps51 is the VFT complex component that binds the N terminus of Tlg1.
Because this region and that of the closest mammalian Tlg1 homologue, syntaxin 6, are not conserved, they hypothesize that the mammalian VFT
complex possesses a divergent subunit.
cells being the most similar to the
wild type (Fig. 2, C and E). The missorting of
vacuolar proteases in VFT complex mutants is not total, a fraction of
those proteins normally reach the vacuole where they are processed and
activated because of the correct acidification of this organelle (46,
47) (Fig. 1B). For this reason a reduced hydrolytic activity
of the vacuole is not a likely explanation for the slower kinetics of
autophagy and pexophagy seen in the vps51
and VFT mutant
strains. Rather, these differences may reflect a slower cellular
metabolism. However, analysis of autophagosomes by electron microscopy
revealed that those structures have a reduced size in
vps51
and vps52
cells indicating that the
membrane expansion process may be impaired (Fig. 3). This is an
interesting finding because it suggests that a specific block in the
Cvt pathway reduces autophagosome size indicating that this route may
be used to transport membranes to the PAS during nitrogen starvation.
Alternatively, the VFT components and Vps51 may play some direct role
in membrane delivery for autophagosome expansion. Further
studies will be needed to fully identify the source
membranes for Cvt vesicles and autophagosomes.
Specific factors required for autophagy, pexophagy, and Cvt pathway
and VFT mutant strains (Fig. 7, C and
D).3 The Aut7
targeting process requires the presence and the correct functioning of
the two conjugating systems, the autophagy-specific PtdIns 3-kinase
complex and Apg9 (20, 21, 24, 76). The proper modification and
localization of Aut7 in the absence of the VFT complex indicates that
all of these Apg/Cvt factors are still fulfilling their functions.
Investigating the cellular distribution of the remaining Apg/Cvt
proteins, we found that in the absence of either the VFT complex or
Tlg2, GFP-Cvt9 is dispersed in several fluorescent dots (Fig. 7,
A and B, data not shown). Cvt9 plays an essential
role in targeting the prApe1-Cvt19 complex to the site of Cvt vesicle
formation (16, 38). In agreement with this model, we found that in the
same cells, prApe1 and Cvt19, together with Cvt9, were no longer able
to reach the PAS (Fig. 7, C and D). The cause of
Cvt9 mislocalization in the VFT mutant cells is still unclear.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. Hugh Pelham, Yoshinori Ohsumi, and Lois Weisman for plasmids and antibodies. We are also very grateful to Drs. Symeon Siniossoglou and Hugh Pelham for communication of results before publication and Dorothy Roak Sorenson for help with electron microscopy.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Public Health Service Grant GM53396 (to D. J. K.) and by a European Molecular Biology Organization long-term fellowship (to F. R.).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: University of
Michigan, Dept. of Molecular, Cellular and Developmental Biology, Ann
Arbor, MI 48109-1048. Tel.: 734-615-6556; Fax: 734-647-0884; E-mail:
klionsky@umich.edu.
Published, JBC Papers in Press, November 20, 2002, DOI 10.1074/jbc.M210436200
2 F. Reggiori and D. J. Klionsky, unpublished observations.
3 J. Guan and D. J. Klionsky, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: Cvt, cytoplasm to vacuole targeting; Apg, autophagy; CFP, cyan fluorescent protein; GFP, green fluorescent protein; HA, hemagglutinin; PA, protein A; PAS, preautophagosomal structure; PtdIns, phosphatidylinositol; tSNARE, target soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor; VFT, Vps fifty-three; vps, vacuolar protein sorting; vSNARE, vesicle SNARE; YFP, yellow fluorescent protein; SMD, synthetic minimal medium; MES, 4-morpholineethanesulfonic acid.
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