SEARCH

SEARCH BY CITATION

Keywords:

  • CORVET;
  • ESCRT;
  • HOPS;
  • overexpression screen;
  • Vps4

Abstract

  1. Top of page
  2. Abstract
  3. Results and Discussion
  4. Conclusions
  5. Materials and Methods
  6. Acknowledgments
  7. References
  8. Supporting Information

A large number of proteins involved in the biogenesis of yeast endosomes and vacuoles have been identified based on screens that scored for inactivation of proteins. Such screens may, however, miss important regulators of the pathway. Here, we present a visual screen in which we examined the effects on vacuole morphology if any of the 6153 yeast open reading frames was overexpressed. Using a progressive screening procedure, we could identify a total of 53 genes. Among the most striking endosomal proteins are the CORVET/HOPS subunits Vps3, Vps18 and Vps39 and the putative tethering inhibitor Ivy1. Furthermore, six endosomal sorting complex related to transport (ESCRT) proteins led to altered vacuole morphology if overproduced. Among the novel proteins, we identify Yer128w as an endosomal protein that interacts with the AAA-ATPase Vps4, and therefore named it Vfa1 (Vps Four-Associated 1). We present evidence on the possible role of these novel proteins in trafficking to the vacuole. Our data provide novel insights into the regulation of protein trafficking.

Transport in the endomembrane system relies on conserved proteins and protein complexes that mediate vesicle budding, membrane remodeling and fusion. Endocytic transport is initiated at the plasma membrane, and directs, for instance activated, and subsequently ubiquitinated cell surface receptors to the endosome for downregulation (1). At the endosome, receptors are sorted into intraluminal vesicles (ILVs) with the help of the endosomal sorting complex related to transport (ESCRT) machinery (2). Once all receptors are cleared off the endosomal surface, the mature multivesicular endosome/body (MVB) fuses with the vacuole/lysosome, which provides the hydrolases required for ILV and protein degradation. The lysosomal hydrolases can reach the vacuole along distinct pathways: either by vesicular transport via the endosome/MVB pathway, by direct transport from the trans-Golgi network (TGN) to the vacuole with the help of adaptor protein complex 3 (AP-3), or by the autophagy-like cytosol-to-vacuole transport pathway (1). Some soluble hydrolases are recognized at the TGN by distinct transmembrane receptors, which release their cargo at the late endosome and are subsequently rerouted back to the TGN with the help of the retromer complex (3). It is thought that retromer-dependent sorting and ESCRT-mediated ILV formation need to be finished before the mature MVB fuses with the vacuole. The fusion of endosomes or vesicles with the vacuole requires the Rab7 GTPase Ypt7 and the HOPS tethering complex for their initial recognition, followed by the SNARE-dependent bilayer mixing (4).

Several regulatory processes accompany the trafficking pathways to the vacuole. Two phospholipids, phosphatidyl-inositol-3-phosphate (PI-3-P) and PI-3,5-P2, serve as membrane-binding platforms for several ESCRT and retromer subunits. PI-kinases and phosphatases therefore play critical roles in endosomal biogenesis (5,6). Likewise, a number of proteins of the endocytic pathway bind ubiquitin or have ubiquitin ligase activity, which is required for efficient sorting of cell surface receptors into ILVs (7). Finally, kinases and phosphatases regulate the activity of the described complexes. One prominent example is the casein kinase Yck3, which phosphorylates the Vps41 subunit of the HOPS complex once it arrives at the vacuole and controls its function in the AP-3 pathway (8–10).

Although most of the trafficking and sorting machinery has been identified based on powerful genetic (deletion) screens, and led to the identification of VPS, END, PEP, VAC and VAM genes (11–15), additional regulators like Yck3 were identified and characterized later (8,16). Likewise, a negative regulator of the ESCRT pathway, Ist1, was missed, because its deletion did not alter ESCRT activity at the first glance (17). We reasoned that overexpression of proteins involved in negative regulation would affect endosomal sorting, whereas their deletion probably would have no visible effect and therefore decided to use an available overexpression library to identify novel factors involved in vacuole biogenesis (18). Here, we present a set of 53 genes that result in altered vacuole morphology and protein trafficking upon overexpression.

Results and Discussion

  1. Top of page
  2. Abstract
  3. Results and Discussion
  4. Conclusions
  5. Materials and Methods
  6. Acknowledgments
  7. References
  8. Supporting Information

A screen for vacuole morphology defects upon gene overexpression

Overexpression of genes can result in significant imbalances in protein complexes by sequestering critical factors. We reasoned that negative regulators of protein transport should affect endosomal and vacuole biogenesis and therefore screened an overexpression library for aberrant vacuole morphology. All genes in this library are encoded by a high-copy 2-µ plasmid and under the control of the strong, inducible GAL1 promoter. Due to their cloning, all genes contain a C-terminal His6-HA-ProteinA cassette, which did not interfere with the functionality of most proteins (19), although we took this possibility into account during our analysis (see below). To identify proteins that could interfere with endosomal and vacuole biogenesis, we induced the expression of plasmid-encoded genes for 24 h at 30°C in rich and nonselective medium (YPG), stained vacuoles with the lipophilic dye FM4-64 and analyzed vacuole morphology by fluorescence microscopy. The plasmid loss was around 10% under these conditions. We then classified vacuole morphology according to previous VPS screens (13,14,16): wild-type cells have one to three normal round vacuoles, class B mutants multiple smaller vacuoles, class C mutants have strongly fragmented vacuoles, class D mutants an enlarged vacuole and class E mutants have an enlarged prevacuolar compartment (Figure 1A). Of the 6153 open reading frames (ORFs), 218 caused altered vacuole morphology. Several ORFs that caused a mild phenotype were eliminated from further analysis. Likewise, proteins with an assigned function at a distinct compartment like the endoplasmic reticulum (ER) were not included. We then scored the 218 ORFs under conditions of plasmid maintenance and deleted ORFs showing mild phenotypes under these conditions. This procedure reduced the list to 53 ORFs (Tables 1–5).

image

Figure 1. Representative vacuolar phenotypes. Overexpression of certain genes affects vacuole morphology. Strain Y258 carrying different overexpression plasmids (pBG1805) was grown in selective medium containing 2% galactose (SGC) to induce overexpression. Vacuoles were stained with FM4-64 as described in Materials and Methods. A) Representative phenotypes obtained in the screen are shown. Indicated phenotypes correspond to following genes: class B, YOL159C-A; class C, IVY1; class D, FRK1; class E, VPS2. B) Examples of genes that affect cell and vacuole morphology when overexpressed. C) Phenotype induced by overexpression of VPS4. Size bar = 10 µm. DIC, differential interference contrast.

Download figure to PowerPoint

Table 1.  Known catalysts in vacuolar trafficking
LocusCommon name(s)Vacuolar phenotype (overexpression)Vacuolar phenotype (deletion)CPY secretionCAN sensitivityLocalizationFunction
  1. Phenotypic characteristics when indicated genes are overexpressed from a 2-µ GAL 1pr-vector. Vacuole phenotypes were scored based on 50–150 cells examined for each strain. The percentage of amorphous cells is given in parenthesis. As a comparison, vacuolar phenotypes of the deletion mutants of vps genes were added according to (1). CPY secretion due to overexpression was estimated visually using a plate assay (see Materials and Methods and Figure S1). Scoring: ‘−’, like wild-type; ‘+’, more secretion than wild-type; “’, less secretion than wild-type. Sensitivity to CAN was estimated by growth on SGC-Arg plates containing 1 µg/mL CAN (see Figure S2). Scoring: ‘−’, like wild-type; ‘+’, more sensitive than wild-type. Information about localization and function of proteins is taken from the yeast genome database (www.yeastgenome.org).

  2. CAN, canavanine; n.d., not determined; PM, plasma membrane.

  3. aNot determined because plasmid could not be isolated from library host strain for further analysis.

ESCRT and related
 YCL008CVPS2310% D, 10% B, 10% EE++EndosomeESCRT-I complex, MVB sorting
 YJR102CVPS2525% D, 5% B (5% amorph)E++EndosomeESCRT-II complex, MVB sorting
 YKL002WVPS2, DID430% EE++EndosomeESCRT-III complex, MVB sorting
 YPL065WVPS2840% D, 10% BE+EndosomeESCRT-I complex, MVB sorting
 YPL084WBRO115% B, 5% DE++EndosomeMVB sorting, Doa4 recruitment
 YPR173CVPS435% E (15% amorph)E+EndosomeAAA-ATPase, ESCRT-III disassembly
HOPS/CORVET
 YDL077CVPS3910% B, 10% CC++VacuoleHOPS complex, vacuole fusion
 YDR495CVPS310% B, 45% C (10% amorph)D++EndosomeCORVET complex
 YLR148WVPS1850% B, 20% D (larger cells)C+VacuoleHOPS/CORVET complex
Vacuolar proteins
 YCR068WATG1575% B n.d.aVacuoleIntravacuolar lipase
 YDR229WIVY155% C +Vacuole 
 YER072WVTC110% B, 10% C +VacuoleVTC complex
 YJL059WYHC3, BTN125% B VacuoleTransport of arginine into vacuole
 YMR297WPRC120% B +VacuoleVacuolar CPY
Other
 YBR164CARL130% B (15% amorph) ++CytosolSoluble GTPase involved in trafficking
 YIL088CAVT720% B (10% amorph) +Vacuole/PMVacuolar amino acid transporter
 YIL173WVTH135% B, 5% C (10% amorph) n.d.aEndosomePutative cargo receptor (Vps 10-homolog)
 YKR014CYPT5220% C, 10% D (25% amorph) EndosomeRab GTPase, endocytosis
 YOR036WPEP12, VPS630% B, 5% DD++EndosomeEndosomal t-SNARE
 YOR212WSTE420% B, 15% D (40% amorph) PMActivation of mating signaling pathway
Table 2.  Protein modification
LocusCommon name(s)Vacuolar phenotype (overexpression)CPY secretionCAN sensitivityLocalizationFunction
  1. Phenotypic characteristics when indicated genes are overexpressed from a 2-µ GAL 1pr-vector. Vacuole phenotypes were scored based on 50–150 cells examined for each strain. The percentage of amorphous cells is given in parenthesis. CPY secretion due to overexpression was estimated visually using a plate assay (see Materials and Methods and Figure S1). Scoring: ‘−’, like wild-type; ‘+’, more secretion than wild-type; ‘’, less secretion than wild-type. Sensitivity to CAN was estimated by growth on SGC-Arg plates containing 1 µg/mL CAN (see Figure S2). Scoring: ‘−’, like wild-type; ‘+’, more sensitive than wild-type. Information about localization and function of proteins is taken from the yeast genome database (www.yeastgenome.org).

  2. CAN, canavanine; n.d., not determined; PM, plasma membrane.

  3. aNot determined due to general growth defect.

  4. bNot determined because plasmid could not be isolated from library host strain for further analysis.

Kinases/Phosphatases
 YDR075WPPH320% B (10% amorph)+Cytosol/nucleusProtein phosphatase complex
 YKL126WYPK110% B+Cytosol/PMSer/Thr protein kinase
 YPL141CFRK195% D+n.d.aCytoplasmPutative kinase
Ubiquitin-modification
 YDR054CCDC3415% B, 20% E+Cytosol/nucleusUbiquitin-conjugating enzyme (E2)
 YDR143CSAN120% B, 10% D (10% amorph)+n.d.aCytosol/nucleusUbiquitin-protein ligase
 YMR275CBUL135% B, 15% Dn.d.bn.d.bPMUbiquitin-binding component of Rsp5
Lipid modification
 YOL003CPFA425% B, 10% DCytosol/nucleusPalmitoyltransferase
Table 3.  Other known catalysts
LocusCommon name(s)Vacuolar phenotype (overexpression)CPY secretionCAN sensitivityLocalizationFunction
  1. Phenotypic characteristics when indicated genes are overexpressed from a 2-µ GAL 1pr-vector. Vacuole phenotypes were scored based on 50–150 cells examined for each strain. The percentage of amorphous cells is given in parenthesis. CPY secretion due to overexpression was estimated visually using a plate assay (see Materials and Methods and Figure S1). Scoring: ‘−’, like wild-type; ‘+’, more secretion than wild-type; ‘’, less secretion than wild-type. Sensitivity to CAN was estimated by growth on SGC-Arg plates containing 1 µg/mL CAN (see Figure S2). Scoring: ‘−’, like wild-type; ‘+’, more sensitive than wild-type. Information about localization and function of proteins is taken from the yeast genome database (www.yeastgenome.org).

  2. CAN, canavanine; n.d., not determined.

  3. aNot determined due to general growth defect.

YBR159WIFA3830% B, 40% C+ERMicrosomal β-keto-reductase
YDR169CSTB340% B, 5% ECytosol/nucleusRRPE-binding protein
YDR525W-ASNA225% B, 10% D (10% amorph)+Cytosol 
YGR109CCLB635% B (10% amorph)n.d.a B-type cyclin
YHR061CGIC140% B, 5% D (40% amorph)n.d.aBud neckInvolved in budding and cellular polarization
YLR227CADY430% B+n.d.aSpindleComponent of meiotic outer plaque
YLR443WECM760% B (10% amorph)+ Integral membrane protein involved in calcium uptake
YNL233WBNI490% B, 5% D (100% amorph)+Bud neckTargeting of Glc7 and chitin synthase III
YOR166CSWT125% BCytosol/NucleusRNA endoribonuclease
Table 4.  Putative proteins of unknown function
LocusVacuolar phenotypeCPY secretionCAN sensitivityCps 1 traffickingSte3 sortingAP-3 transportLocalization
  1. Phenotypic characteristics when indicated genes are overexpressed from a 2-µ GAL1pr-vector. Vacuole phenotypes were scored based on 50–150 cells examined for each strain. The percentage of amorphous cells is given in parenthesis. CPY secretion due to overexpression was estimated visually using a plate assay (see Materials and Methods and Figure S1). Scoring: ‘−’, like wild-type; ‘+’, more secretion than wild-type; ‘’, less secretion than wild-type. Sensitivity to CAN was estimated by growth on SGC-Arg plates containing 1 µg/mL CAN (see Figure S2). Scoring: ‘−’, like wild-type; ‘+’, more sensitive than wild-type. Trafficking of GFP-Cps1, Ste3-GFP and GNS (for AP-3 transport) was estimated by fluorescence microscopy as described in Materials and Methods. ‘−’, localization of the marker-like wild-type; ‘+’, marker is mislocalized. Localization based on GFP-tagged proteins (see Figure 3A,B and Materials and Methods).

  2. CAN, canavanine; n.d., not determined.

  3. aNot determined due to general growth defect.

YBR071W20% B (25% amorph)++Bud neck/cytosol
YDR514C50% B, 10% D+Mitochondrion
YDR541C30% D++++Cytosol
YER128W30% B, 20% D+++Endosome, cytosol
YGR204C-A15% B, 20% C++ 
YHR112C40% B (10% amorph)++Nucleus/cytosol
YIL089W20% B, 5% D+++ER/vacuole
YKL222C25% B, 15% C++Nucleus/spindle
YKR041W30% B, 10% E (35% amorph)++Nucleus/spindle
YOL159C-A50% B+Nucleus/cytosol
YOR223W10% B, 10% Dn.d.aER/vacuole
Table 5.  Orphan ORFs
LocusVacuolar phenotypeCPY secretionCAN sensitivity
  1. Phenotypic characteristics when indicated genes are overexpressed from a 2-µ GAL1pr-vector. Vacuole phenotypes were scored based on 50–150 cells examined for each strain. The percentage of amorphous cells is given in parenthesis. CPY secretion due to overexpression was estimated visually using a plate assay (see Materials and Methods and Figure S1). Scoring: ‘−’, like wild-type; ‘’, less secretion than wild-type. Sensitivity to CAN was estimated by growth on SGC-Arg plates containing 1 µg/mL CAN (see Figure S2). Scoring: ‘−’, like wild-type; ‘+’, more sensitive than wild-type.

  2. CAN, canavanine.

YBL094C15% B, 5% D+
YDL162C35% B (5% amorph)
YFL030C-A25% D, 5% B (5% amorph)
YGL052W30% D, 10% B
YOR309C30% B, 30% B
YOR314W20% B, 10% D

To validate our screen, we compared our candidates to known factors that caused vacuole fragmentation upon overproduction. The CORVET subunit Vps3 is believed to interfere with the biogenesis of the vacuolar HOPS tethering complex if present in excess (20). Vps3 was indeed among the isolated candidates. Likewise, Ivy1 was identified in our screen. This protein interacts with the HOPS subunit Vps33 and the Rab Ypt7, and is induced during stationary phase (21). The re-identification of Vps3 and Ivy1 indicates that (i) our screen was indeed able to identify endosomal factors and (ii) that the C-terminal tags generally did not inactivate the proteins. In addition to these factors, we could identify several genes that interfered with cell (and vacuole) morphology, in good agreement with a previous study (19) (Figure 1B).

During the course of our analysis, we noticed that several transmembrane proteins of the ER and Golgi affected vacuole morphology, presumably due to spill-over from the resident compartment and subsequent sorting into the endocytic pathway. We therefore did not include most membrane proteins with multiple transmembrane domains in our further analysis, although we are aware that we might miss out on potential regulators. The remaining transmembrane proteins in our list may have similar side effects on trafficking.

To further validate our results, we employed two distinct assays. VPS proteins were initially identified in screens that scored for the secretion of the vacuolar hydrolase carboxypeptidase Y (CPY). We thus measured CPY secretion upon overexpression, taking the BY wild-type strain as a control. In contrast to the corresponding wild-type employed in the VPS screen (13), BY wild-type cells secrete some CPY (Figure S1). Consequently, we were able to observe both suppression of secretion and its enhancement due to gene overexpression (Tables 1–5 and Figure S1). The reduction in CPY secretion is possibly due to interference with general secretion or more efficient recycling of the cargo receptor Vps10. The exact reason for this phenomenon is not yet known. As a second assay, we employed sensitivity to the toxic arginine homolog canavanine, which is transported into cells via the arginine permease Can1. Can1 normally cycles between endosomes and the plasma membrane and therefore leaves wild-type cells resistant to canavanine, whereas ESCRT deletions, for instance, result in increased sensitivity due to the stabilization of the permease at the plasma membrane (22). Indeed, several overexpressors-induced canavanine sensitivity (Figure S2). Below, we have grouped the different genes according to their known function and evaluated the implications in the context of the literature.

Identification of endosomal proteins that affect vacuole biogenesis

Our main interest of the screen was the identification of endosomal and vacuolar proteins with a putative regulatory function in trafficking. Indeed, we could identify three proteins of the HOPS and CORVET complex, several ESCRT subunits (presented below) and 10 endosomal and vacuolar proteins, which reproducibly affected vacuole morphology upon overexpression (Table 1). Besides Vps3 and Ivy1, we isolated the HOPS subunit Vps39 and Vps18. In previous studies, we observed that overproduction of chromosomally integrated VPS39 interfered with vacuole morphology, while overproduction of chromosomally integrated VPS18 did not (23,24), indicating that this effect is dose dependent. Interestingly, other HOPS and CORVET subunits were not identified, suggesting a more dynamic association of Vps39, Vps18 and Vps3 with their complexes, as previously demonstrated for Vps3 (20).

Seven of the characterized endosomal proteins were probably identified due to their general negative impact on the protein sorting machinery. This includes Atg15, a transmembrane protein with lipase activity found in the vacuole lumen (25); Avt7, a multispan amino acid transporter of the vacuole (26); Btn1, an arginine transporter that has been implicated in acidification of the yeast vacuole (27); Pep12, the endosomal syntaxin homolog involved in transport between Golgi and endosomes (28); Vtc1, a subunit of the Vtc complex of polyphosphate phosphatases implicated in vacuole fusion and microautophagy (29,30); Vth1, a Vps10-like transporter (31) and Prc1 (CPY) (32), which is transported to the vacuole via the Vps10 cargo receptor (33). CPY is known to accumulate in the culture supernatant, if provided in excess.

We also identified three proteins related to GTPase-dependent reactions. The Arf-like Arl1 protein (34,35) is involved at the endosome–TGN interface and required for the GGA-dependent transport to the vacuole. Arl1 overproduction results in increased canavanine sensitivity (Figure S2), consistent with a defect in Can1 recycling at the endosome. Furthermore, we identified Ste4, which affects both cell and vacuole morphology upon overproduction (Figure 1B). Ste4 is the β-subunit of the heterotrimeric G-protein complex, which activates together with the γ-subunit Ste18 the mating signaling pathway (36). We assume that the excess of Ste4 displaces its α-subunit Gpa1, which may subsequently traffic to endosomes and perturb its biogenesis by binding and activating Vps34 (37). Finally, Ypt52 was identified, which is homologous to Rab5 GTPase in yeast, Vps21/Ypt51 and Ypt53 (38). Its function in yeast is still uncertain, even though it binds like Vps21 to the CORVET subunit Vps8 and accumulates like many endosomal proteins in enlarged class E endosomes (39) (our unpublished observations). We assume that the overexpressed Rab is not prenylated due to its C-terminal tags, and thus generate an inactive, soluble Rab, which could interfere with endosomal function.

Proteins involved in posttranslational modifications

Within our screen, we expected to find kinases and phosphatases that may target proteins involved in endosomal biogenesis (Table 2). Indeed, two kinases (Ypk1 and Frk1) and one phosphatase (Pph3) were identified. Among these, Pph3 is an interesting candidate as its overexpression results in a strong sensitivity to canavanine and suppression of CPY secretion. Pph3 is part of a phosphatase complex with multiple functions in cell division, DNA repair and Tor signaling (40–43).

Overexpression of the E2-enzyme Cdc34, the Rsp5 interacting protein Bul1 and the ubiquitin ligase San1 also resulted in alterations in vacuole morphology, as did the overexpression of the acyltransferase Pfa4, a polytropic membrane protein. The relevance of these factors is not yet clear as only CDC34 and SAN1 overexpression resulted in a weak CPY secretion phenotype. Table 3 lists additional factors identified in our screen, which we do not take into account as endosomal regulators, because they have assigned functions elsewhere in the cell. For instance, BNI4 overproduction results in completely amorphous cells (Table 3 and Figure 1B), consistent with earlier observations (19). Therefore, the CPY secretion phenotype in these cells is probably due to cell lysis.

Several ESCRT proteins affect vacuole biogenesis

One striking observation was the identification of several ESCRT proteins that affected vacuole morphology upon overproduction (Table 1). We did not observe any preference for one of the four complexes, and found Vps23 and 28 (ESCRT-I), Vps25 (ESCRT-II), Vps2 (ESCRT-III) and Vps4. Some of the overexpression phenotypes may be due to the C-terminal tagging, although we would expect a class E-like phenotype under these conditions. This was only the case for VPS4 and VPS2 overexpression (Table 1). In addition, Vps4 overproduction resulted in FM4-64 positive dots that were present throughout the cytoplasm (Figure 1C). These dots might represent endosomal intermediates, which would indicate that the endocytic pathway is slowed down under these conditions. Furthermore, two hallmarks of ESCRT deletions, sensitivity to canavanine (Figure S2) and missorting of Cps1 (not shown) were also observed for ESCRT overexpressors. Almost all of these subunits also promoted CPY secretion (Table 1 and Figure S1). These observations are similar to the findings on Ist1, a negative regulator of Vps4 function, which also resulted in additional ESCRT-specific phenotypes like missorting of green fluorescent protein (GFP)-Cps1 upon overexpression (17) (M. Babst, personal communication). In addition to the described ESCRT subunits, 12 other genes caused canavanine sensitivity upon overexpression. Among these, three previously uncharacterized ORFs showed strong defects comparable to ESCRT overexpression: YDR541C, YIL089W and YER128W (Table 4). Deletion of these genes did not cause a GFP-Cps1 trafficking defect, which would be expected for negative regulators of ESCRT function (see below).

New players in trafficking to the vacuole

In addition to proteins with known function, the screen revealed several proteins of unknown function, which might have a role in trafficking to the vacuole (Table 4) and some ambiguous ORFs (Table 5). To unravel novel players in trafficking to the vacuole, we focused on the uncharacterized proteins (Table 4). We first analyzed, which trafficking routes to the vacuole were affected by overproduction. We looked for defects in localization of Ste3-GFP and GFP-CPS as model cargoes of the endocytic pathway, and GNS (GFP-Snc1-TMD-Nyv1)(44) as a cargo of the AP-3 pathway (Figure 2). This analysis enabled us to arrange some of the uncharacterized genes into groups that share certain phenotypes and therefore might act in one trafficking pathway as discussed below. We also analyzed the deletion mutants of these genes in the same trafficking assays, but could not detect clear mislocalization of the marker proteins (Figure S3A). However, we detected a weak mislocalization of Ste3-GFP to the ER in some deletion mutants (Figure S3A). The deletion mutants also showed no increased sensitivity to canavanine or abnormal CPY secretion (not shown).

image

Figure 2. Phenotypic characteristics caused by overexpression of uncharacterized genes. Eleven uncharacterized genes identified in the screen (Table 4) were analyzed for trafficking defects to the yeast vacuole. A) Wild-type and indicated deletion strains were grown in synthetic medium to retain empty pRS426 plasmids and CEN plasmids containing trafficking markers (pCU1775, GFP-CPS; pCU2944, GNS; pCU3078, STE3-GFP) as control. Overexpression was induced by addition of 2% galactose and incubation for 24 h followed by observation under the microscope. B–D) Cells carrying indicated marker plasmids and 2-µ plasmids for overexpression of indicated genes were grown as described in (A). Size bar = 5 µm. DIC, differential interference contrast.

Download figure to PowerPoint

The overexpression of YBR071W, YGR204C-A, YHR112C and YKL222C resulted in an accumulation of the endocytic cargo Ste3 in the cell, without affecting sorting of the biosynthetic cargo Cps1 (Figure 2B), which indicates that these proteins might have a role at the early endosome or during endocytosis.

The genes YDR541C, YER128W and YIL089W share an accumulation of GFP-CPS at prevacuolar compartments and on the vacuolar membrane, as well as increased canavanine sensitivity when overexpressed (Figures 2C and S1). In addition, overexpression of these genes causes CPY secretion (Figure S1). Interestingly, overexpression of all of these genes triggers a partial class D-like vacuole morphology, which was also observed for some ESCRT subunits (Table 1), supporting the idea that these proteins affect endosomal maturation or ESCRT activity.

YDR514C, YKR041W, YOL159C-A and YOR223W showed no missorting phenotype of either model cargo (Figure 2D). Therefore, it remains unclear if these proteins have a specific role in trafficking.

None of the overexpressors affected the AP-3 pathway (Figures 2 and S3A), which can be explained by the fact that even deletions of essential AP-3 coat proteins have wild-type vacuoles (10).

In addition to the trafficking assays described above, we tried to investigate effects on the Cvt pathway by following the clipping of Ape1 to its mature form due to fusion of Cvt vesicles with the vacuole (45). While we were able to detect a weak clipping defect for the deletion of YKR041W (Figure S3B), we were unable to carry out the assay under overexpression conditions due to the increased level of proteinA-tagged protein that interfered with the western blot detection of Ape1 (not shown).

To further investigate the role of the newly identified candidates, we localized these proteins by C-terminal GFP-tagging (Figure 3A–E). We observed an unexpected variety of localizations, which makes a common assignment of their interfering function in vacuole biogenesis challenging. Yhr112c and Yol159c-a localize to the nucleus and to the cytoplasm. Ykr041w and Ykl222c also localize to the nucleus and the mitotic spindle (Figure 3A). Ydr514c, however, clearly localized to mitochondria (Figure 3B). Yil089w and Yor223w were found at the ER and partially in the vacuolar lumen (Figure 3C). These two proteins have predicted transmembrane domains (http://www.yeastgenome.org). We noticed though that even strong overexpression of YIL089W from the GPD promoter does not lead to mislocalization of this protein to other compartments (Figure 3F). Ybr071w was found in the cytosol and at the bud neck (Figure 3D). This agrees with the high percentage of amorphous cells with elongated buds observed, when this gene was overproduced (not shown). Overexpression probably interferes with septin ring formation or actomyosin ring contraction. GFP-tagged Ydr541c and Ygr204c-a could not be detected if expressed from a plasmid but overexpressed Ydr541c was located in the cytoplasm (Figure 3F).

image

Figure 3. Localization of uncharacterized proteins identified in the screen and effect of untagged protein overexpression. A–E) Localization of uncharacterized proteins identified in the overexpression screen. Expression of C-terminally tagged proteins from a CEN plasmid under control of the NOP1pr in wild-type strain. Cells were grown in synthetic medium to retain plasmids. F) Localization of Ydr541c, Yer128w and Yil089w by GFP-tagging in the genome. Growth in YPD followed by washing in synthetic medium to reduce background in the GFP channel. G) Yeast strains overexpressing YDR541C, YER128W or YIL089W from the strong GPD promoter were transformed with plasmid pCU3078 to analyze Ste3 trafficking or Cps1 was N-terminally tagged with PHO5pr-GFP to analyze biosynthetic transport. Size bar = 5 µm. DIC, differential interference contrast.

Download figure to PowerPoint

Interestingly, the protein Yer128w was detected in the cytoplasm and in dots proximal to the vacuole, even if expressed from the endogenous promoter (Figure 3A,F; see below).

As overexpression of YER128W, YDR541C and YIL089W showed the strongest influence on trafficking of GFP-CPS (Figure 2C and Table 4), we asked whether missorting phenotypes could be reproduced for overexpression of the untagged proteins under control of the strong GPD promoter. For YDR541C, we could reproduce the defects, whereas overproduction of the other two proteins was without effect (Figure 3G). At present, we cannot yet distinguish if this wild-type phenotype is due to the reduced overproduction or the removal of the epitope tags.

The novel endosomal protein Vfa1/Yer128w interacts with Vps4

We then decided to explore the role of Yer128w in more detail. To address whether Yer128w localizes to endosomes, we performed colocalization studies with the endosomal Rab GTPase Vps21, the late endosomal/vacuolar Rab Ypt7 and Mnn9 as a resident protein of the Golgi (23,39). Yer128w clearly colocalizes with Vps21-positive structures and partially to Ypt7-positive structures, but not with Mnn9 (Figure 4A). Furthermore, previous high-throughput analyses identified Yer128w as a Vps4 interactor (46,47). We thus tested this physical interaction directly using a pull-down approach with purified Vps4 and observed a specific interaction (Figure 4B). We therefore named YER128W VFA1 for Vps Four-Associated 1.

image

Figure 4. The endosomal protein Vfa1/Yer128w interacts with Vps4. A) Yeast with chromosomally GFP-tagged Vfa1 (YER128W) carrying plasmids encoding red fluorescent protein (RFP)-VPS21 or RFP-YPT7 as early and late endosomal markers or integrated MNN9-TOMATO as Golgi marker were grown in synthetic medium and analyzed by fluorescence microscopy. Colocalization of Vps21 and Yer128w/Vfa1 are indicated with arrows. B) Localization of Vfa1 depends on Vps4. Wild-type or vps4 mutant cells were transformed with plasmids encoding NOP1pr-VFA1-eGFP. Growth in synthetic medium, FM4-64 staining as described in Materials and Methods. C) Vfa1 interacts with Vps4. Recombinant His6-Vps4 or buffer was added to Ni-NTA beads. Yeast lysate of a strain with C-terminal TAP-tagged VFA1 was used for Ni-NTA pull down. Retained proteins were analyzed by western blot with a PAP or anti-His antibody. A and C) Size bar = 5 µm.

Download figure to PowerPoint

Another Vps4-interacting protein, Ist1, also localizes to the class E compartment, when VPS4 was deleted (17). When we analyzed the Vfa1 in vps4 mutant cells, we could not detect any dot-like localization, although the class E compartment was clearly visible (Figure 4C). This indicates that Vfa1 requires Vps4 for its recruitment to endosomes.

Conclusions

  1. Top of page
  2. Abstract
  3. Results and Discussion
  4. Conclusions
  5. Materials and Methods
  6. Acknowledgments
  7. References
  8. Supporting Information

Our overexpression screen on vacuole morphology provides novel insights into the regulation of the endocytic pathway. We could re-identify two known factors, the CORVET subunit Vps3 and the putative regulator Ivy1, which both cause massive vacuole fragmentation. Both are of immediate interest for future research as both likely interfere with functionality of the HOPS and CORVET complexes (20,21,39). In addition to these, our data provide novel insights into ESCRT-mediated regulation at the endosome. We found that several ESCRT subunits interfered with endosomal biogenesis. This is consistent with our recent analysis of Ypt7-induced modulation of the vacuole membrane (23). Indeed, overproduction of the ESCRT-III subunit Vps2 caused the expected class E phenotype. Surprisingly, the class E phenotype was not the general outcome, when ESCRTs were overproduced. For instance, Vps28 overproduction results in a class D phenotype, which is observed if the endosomal fusion machinery is impaired. Potentially, Vps28 contacts the fusion machinery to coordinate endosomal protein sorting with membrane fusion. Similar functions might apply to other subunits. However, we are aware that many ESCRT subunits do not tolerate large C-terminal tags, and may thus act as dominant negatives in our screen. Future studies therefore need to address the relevance of our findings.

Our screen identified several uncharacterized ORFs, some of which strongly affect sensitivity to canavanine, sorting of Cps1 and CPY secretion (YDR541C, YER128W and YIL089W), similar to regulators of the ESCRT pathway. Both YDR541C and YER128W/VFA1 interact with ESCRT components based on large throughput screens (46,47). Genetic correlation analyses suggest links of YIL089W to the sphingolipid and phospholipid biosynthesis pathways (48). One of the challenges with most of our novel genes is their localization. We so far cannot clearly link their interference with vacuolar biogenesis to their localization in the cell. For instance, Yil089w and Yor223w are ER proteins, and Ykl222c is found on the mitotic spindle. Some proteins may exhibit dual localizations or specific links to the vacuole, like the known ER–nuclear and vacuole–nuclear junctions (49), although we did not address these contact zones in this study. Similarly, the interference of the mitochondrial Ydr514c protein with vacuole biogenesis remains unresolved. While we acknowledge that some of our phenotypes may be non-specific and that tags on the overexpressed proteins provide additional challenges to interpret some of our phenotypes, the selective interference with distinct trafficking pathways suggest that at least some of our identified proteins are linked to novel regulatory circuits in the endosomal system.

This is clearly the case for Yer128w/Vfa1. In addition to the re-identification of Vps3 and Ivy1 and the ESCRTs, Vfa1 is candidate that may fulfill the role of a negative regulator within the endocytic pathway. Vfa1 localizes to endosomes in a Vps4-dependent manner, interferes with biosynthetic sorting to the vacuole and binds directly to Vps4. It behaves similarly to Ist1, which was also identified as a negative Vps4 regulator (17), and could be a valuable protein to dissect the function of Vps4 and ESCRTs at the late endosome.

Like other genetic screens, we might have missed potential regulators. For instance, overproduction of guanine nucleotide activator protein of the Ypt7 Rab GTPase, Gyp7, results in vacuole fragmentation (9,50,51) and was not identified here. However, the screen uncovered an additional approach to identify novel factors operating in the endosomal pathway.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Results and Discussion
  4. Conclusions
  5. Materials and Methods
  6. Acknowledgments
  7. References
  8. Supporting Information

Yeast strains and plasmids

All strains and plasmids used in this study are listed in Table S1. A library containing 6153 ORFs on 2-µ plasmids (pBG1805-GAL1pr-ORFx) in Saccharomyces cerevisiae strain Y258 was purchased from Open Biosystems Inc. Analysis of CPY secretion and canavanine sensitivity assays were carried out in CUY5669, which is isogenic to BY4741 suc2Δ with LEU2 integrated pBHY11 (52). Deletion and genomic tagging were carried out by integration of marker cassettes using standard procedures (53). For localization of uncharacterized proteins, genes were amplified from BY4727 genomic DNA and cloned into pJET1 plasmids (Fermentas), followed by subcloning into BamHI/SacII or NotI/SacII restriction sites of pCU2808. VPS4 was amplified from genomic DNA and cloned into BamHI/HindIII sites of pET32H resulting in pCU284.

Overexpression screen

Cells were inoculated from synthetic medium containing 2% glucose (SDC) and essential amino acids to retain plasmids into 150 µL yeast extract-peptone-galactose medium (YPG) (96 well plates) and incubated for 12–16 h at 30°C. Sterile glass beads were added to each well and cells were grown for another 5 h on a rotary shaker. Beads were removed and cells were stained with FM4-64 (see FM4-64 staining of yeast vacuoles). Vacuolar morphology was analyzed by fluorescence microscopy. Strains that showed altered vacuolar morphology were screened again in 500 µL YPG cultures (1.5 mL tubes). The resulting 218 strains were grown in synthetic medium containing 2% raffinose (SRC), and overexpression was induced by addition of 2% galactose. Cells were grown for 24 h in galactose containing medium, stained with FM4-64 and analyzed under the microscope.

CPY secretion

CPY secretion assay was carried out as described (52). Briefly, strain CUY5669 was transformed with pBG1805 overexpression plasmids. Transformants were picked from SDC-URA plates, resuspended in 5 µL water, and spotted onto synthetic medium with 2% galactose (SGC) to induce overexpression. Cells were grown for 48 h at 30°C, and secretion of CPY was measured by overlaying the agar plate with developer solution (52). Secretion phenotypes were scored by comparing mean intensity values of each yeast spot to wild-type.

Canavanine assay

Yeast strain CUY5669 carrying different pBG1805 overexpression plasmids was grown in SRC-URA overnight at 30°C. Serial dilutions of logarithmically growing cells were plated onto SGC medium without uracil and arginine and indicated canavanine concentrations. Plates were imaged after 4 days of growth at 30°C.

FM4-64 staining of yeast vacuoles

FM4-64 (10 µm) was added to logarithmically growing cells for 30 min (pulse), followed by a washing step with medium and a 2 h chase. All steps were carried out at 30°C.

His6-Vps4 Ni-NTA pull down from yeast lysate

Recombinant His6-VPS4 was purified from Escherichia coli using standard techniques (54). Yeast lysate of strain CUY6444 was prepared by glass bead lysis in 150 mm NaCl, 50 mm Hepes pH 7.4, 0.15% Igepal and 1 mm MgCl2. Three hundred micrograms of His6-Vps4 or buffer were added to Ni-NTA-agarose beads (Quiagen) and incubated for 1 h at 4°C, followed by washing with buffer. Yeast lysate was added to the beads and incubated for 1 h at 4°C in the presence of 20 mm imidazole, followed by washing with buffer (with 20 mm imidazole) and elution in 1 × SDS sample buffer. Eluted proteins were analyzed by western blot using peroxidase anti-peroxidase (PAP) (Sigma) and anti-His antibodies (Quiagen).

Fluorescence microscopy

Cells were stained with FM4-64, washed once with 1 mL PBS buffer or synthetic medium, and mounted on a cover slide for microscopy. Images were acquired using a Leica DM5500 or Leica DM400B microscope (Leica) with a SPOT Pursuit-XS (Diagnostic Instruments) or a DFC350FX (Leica) camera, using filters for RFP, GFP and FM4-64. Pictures were processed using Adobe Photoshop 10 (Adobe Systems) and ImageJ (developed by Wayne Rasband; National Institutes of Health).

Acknowledgments

  1. Top of page
  2. Abstract
  3. Results and Discussion
  4. Conclusions
  5. Materials and Methods
  6. Acknowledgments
  7. References
  8. Supporting Information

We thank Rabea Bartoelke, Franziska Kaplan and Thomas Merkel for their contributions to the screen, Michael Thumm (University of Göttingen, Germany) for the Ape1 antibody and Markus Babst (University of Utah) for plasmids and discussions. This work was supported by the DFG (SFB 431 and 944) and the Hans-Mühlenhoff-foundation (to C. U.).

References

  1. Top of page
  2. Abstract
  3. Results and Discussion
  4. Conclusions
  5. Materials and Methods
  6. Acknowledgments
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Results and Discussion
  4. Conclusions
  5. Materials and Methods
  6. Acknowledgments
  7. References
  8. Supporting Information

Supporting Information

Additional Supporting Information may be found in the online version of this article:

Figure S1: CPY secretion upon gene overexpression. Cells carrying overexpression plasmids encoding the indicated genes were spotted onto SGC-URA plates and CPY secretion was assayed as described in Materials and Methods. Cells that strongly secrete the CPY-invertase chimera turn black and cells that secrete less than wild-type remain white. PET10 was excluded from further analyses.

Figure S2: Canavanine sensitivity upon gene overexpression. Serial dilutions of cells carrying overexpression plasmids encoding the indicated genes were spotted onto SGC-URA-ARG plates containing indicated canavanine concentrations. Plates were scanned after 4 days of growth at 30°C.

Figure S3: Phenotypic characteristics of uncharacterized gene deletions. A) Indicated yeast strains carrying CEN plasmids with indicated markers were grown in synthetic medium, followed by fluorescence microscopy. Size bar = 5 µm. B) Functionality of Cvt pathway upon gene deletion. Indicated yeast strains were grown to logarithmic phase, equal amounts of cells were harvested and lysed by addition of 10% trichloroacetic acid. Maturation of Ape1 was analyzed by western blot using an anti-Ape1 antibody.

Table S1: Yeast strains and plasmids used in this study.

FilenameFormatSizeDescription
TRA_1252_sm_fs1_fs3.pdf5094KSupporting info item
TRA_1252_sm_ts1.pdf88KSupporting info item

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.