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Cell Biology International (2007) 31, 1237–1244 (Printed in Great Britain)
Prevacuolar compartment morphology in vps mutants of Saccharomyces cerevisiae
Jamie M. Hedmana, Matthew D. Egglestonb, Amanda L. Attrydeb and Pamela A. Marshallab*
aDepartment of Biology, State University of New York, College at Fredonia, Fredonia, NY, USA
bDepartment of Integrated Natural Sciences, Arizona State University at the West Campus, MC2352, P.O. Box 37100, Phoenix, AZ 85069, USA


Abstract

Over 60 genes have been identified that affect protein sorting to the lysosome-like vacuole in Saccharomyces cerevisiae. Cells with mutations in these vacuolar protein sorting (vps) genes fall into seven general classes based upon their vacuolar morpholology. Class A mutants have a morphologically wild type vacuole, while Class B mutants have a fragmented vacuole. There is no discernable vacuolar structure in Class C mutants. Class D mutants have a slightly enlarged vacuole, but Class E mutants have a normal looking vacuole with an enlarged prevacuolar compartment (PVC), which is analogous to the mammalian late endosome. Class F mutants have a wild type appearing vacuole as well as fragmented vacuolar structures. vps mutants have also been found with a tubulo-vesicular vacuole structure. vps mutant morphology is pertinent, as mutants of the same class may work together and/or have a block in the same general step in the vacuolar protein sorting pathway. We probed PVC morphology and location microscopically in live cells of several null vps mutants using a GFP fusion protein of Nhx1p, an Na+/H+ exchanger normally localized to the PVC. We show that cell strains deleted for VPS proteins that have been previously shown to work together, regardless of VPS Class, have the same PVC morphology. Cell strains lacking VPS genes that have not been implicated in the same pathway show different PVC morphologies, even if the mutant strains are in the same VPS Class. These new studies indicate that PVC morphology is another tier of classification that may more accurately identify proteins that function together in vacuolar protein sorting than the original vps mutation classes.


Keywords: Saccharomyces cerevisiae, Yeast, Prevacuolar compartment, Late endosome, Morphology, vps Mutants.

*Corresponding author. Department of Integrated Natural Sciences, Arizona State University at the West Campus, MC2352, P.O. Box 37100, Phoenix, AZ 85069, USA. Tel.: +1 602 543 6143; fax: +1 602 543 6073.


1 Introduction

The compartmental nature of eukaryotic cells requires that they contain an efficient mechanism for transporting proteins to and from various intracellular locations. These delivery systems must have a high level of specificity in order to maintain the unique protein composition found in all organelles. The specificity of protein sorting to the lysosome-like vacuole of bakers' yeast, Saccharomyces cerevisiae, is no exception. Unique cellular components must recognize vacuolar proteins, sort them away from secretory proteins, package them into transport vesicles, and deliver them to the vacuole via a prevacuolar compartment (PVC, equivalent to the mammalian late endosome). Many of the molecular mechanisms involved in these delivery pathways are not understood. Several genetic selections have uncovered a large set of yeast mutants that are defective in the vacuolar protein delivery pathway. Most of the gene products affected in these vps (vacuolar protein sorting) mutants are part of the trans-acting cellular machinery that is required for sorting proteins to the vacuole. However, a few mutants in vps proteins have a less direct effect on protein sorting to the yeast vacuole; these include mutants that are involved in the maintenance of the protein composition of the PVC, by serving in the recycling of proteins via vesicle intermediates from the PVC back to the Golgi (Bowers and Stevens, 2005). Nonetheless, all of the vps mutants identified have an aberrantly functioning vacuole, and it is crucial to understand the molecular basis for this defective organelle in the different vps mutants.

The yeast vacuole, which is the equivalent of the mammalian lysosome, is an acidic compartment involved in macromolecular turnover (Klionsky et al., 1990). Vacuolar hydrolases transit the early stages of the secretory pathway en route to the vacuole/lysosome. In a late Golgi compartment, these proteins are actively sorted away from the pool of secretory proteins and are targeted to the vacuole/lysosome directly or via a PVC (endosomal) intermediate, depending on the proteinaceous cargo. For many vacuolar proteases, the arrival in the vacuole is followed by a proteolytic processing event that activates the hydrolase (Bryant and Stevens, 1998; Klionsky et al., 1990; Vida et al., 1993).

Vacuolar proteases transit the early part of the secretory pathway until they reach the late Golgi where they are actively sorted away from the bulk of the proteins, destined for secretion (Vida et al., 1993). Alteration of the vacuolar sorting of several proteases results in its appearance at the cell surface (Bankaitis et al., 1986; Robinson et al., 1988; Rothman et al., 1989; Rothman and Stevens, 1986). This mislocalization and subsequent secretion of vacuolar proteins are the basis of several genetic selections that have resulted in the isolation of a large number of mutants defining over 40 complementation groups specifically defective in the delivery of proteins to the vacuole (Robinson et al., 1988; Rothman and Stevens, 1986). In addition, other selections and screens have identified additional mutants that are defective in vacuolar protein sorting (Avaro et al., 2002; Bonangelino et al., 2002; Entian et al., 1999). Together these mutant collections describe over 60 different proteins involved in the VPS pathway (Bowers and Stevens, 2005).

In order to gain a better understanding of the cellular processes affected in the vacuolar protein sorting mutants, morphological studies of the vacuole were carried out. The vps mutants were found to fall into seven morphological classes, Classes A–F and mutants with tubulo-vesicular vacuoles (Banta et al., 1988; Conibear et al., 2003; Horazdovsky et al., 1994; Kucharczyk and Rytka, 2001; Raymond et al., 1992). Class A mutants contain a wild type appearing vacuole, which consisted of 1–3 prominent structures, which in some cases were interconnected. Class A mutants, therefore, appear to affect the delivery of soluble vacuolar proteins and have little effect on the delivery of membrane to the vacuole. The other classes of vps mutants also have a vacuole that functions at less than wild type capacity and show more severe and distinct morphological defects than Class A mutants. The mutants that comprise the Class B morphology group lack any large vacuolar structure. Instead, these mutants contain about 30–40 small vacuole-like structures. These small structures are acidic, but are not completely competent as targets for the vacuolar protein sorting system. Class C vps mutants show the most pronounced morphological defect; they have no identifiable vacuolar structure but do accumulate many abnormal membrane enclosed structures. Class D mutants are characterized by the presence of a slightly enlarged vacuole structure, defects in mother to daughter vacuole inheritance, and an improperly assembled vacuolar H+-ATPase. Class E vps mutants accumulate a novel organelle distinct from the vacuole. This structure is smaller than the vacuole, is acidic, and contains vacuolar ATPase but not the vacuolar membrane protein alkaline phosphatase (ALP). The Class E structure is thought to represent an exaggerated form of the PVC. Like the Class B mutants, Class F mutants contain small fragmented vacuole-like structures, but they also contain a large vacuole as well. Tubulo-vesicular vacuoles, different in structure than the original morphological classes, have also been seen in several vps mutants (Conibear et al., 2003; Conibear and Stevens, 2000).

This initial characterization of the vps mutants has had its limitations, however. Several vps mutant classes describe a set of trans-activating factors that function at the same point in the lysosomal protein targeting pathway, such as the Class C VPS proteins and subsets of Class E proteins (Bowers and Stevens, 2005). However, the Class A VPS proteins function at several disparate places in the vacuolar pathway. For example, Vps30p functions in autophagy and protein recycling from the PVC to the Golgi (Kametaka et al., 1998; Kihara et al., 2001; Seaman et al., 1997). Vps29p and Vps35p function with the Class B proteins Vps5p and Vps17p and a Class F protein Vps26p to recycle CPY receptor from the PVC to the Golgi, in a protein complex termed the retromer (Seaman et al., 1998). Vps55p functions to traffic proteins from the PVC to the vacuole and also has a defect in endocytosis (Belgareh-Touzé et al., 2002). We feel that the initial classification of the VPS proteins placed many into apparent functional categories that turned out to be incorrect or simplistic for revealing the myriad of pathways of protein sorting to the yeast vacuole.

The focus of this research is to begin to extend this initial classification by studying the structure and subcellular location of the PVC in several vps mutants, using a GFP fusion protein of a bona fide PVC protein, Nhx1p, which is an Na+/H+ exchanger on the PVC membrane (Nass and Rao, 1998). The PVC of S. cerevisiae is a membrane-bound compartment, usually juxtaposed next to the vacuole (Gerrard et al., 2000; Odorizzi et al., 2003; Piper et al., 1995). One of the main functions of the PVC is as a last protein sorting point before the proteins are either sent onto the vacuole or recycled back into the early secretory pathway, reviewed in Bowers and Stevens (2005). This organelle is characterized by its ion balance, and this ion balance is required for proper vacuolar protein sorting out of the PVC (Bowers et al., 2000; Brett et al., 2005). One ion transporter that is required for PVC ion balance is the Na+(K+)/H+ exchanger, Nhx1p (Bowers et al., 2000; Brett et al., 2005; Nass and Rao, 1998). This sodium/proton exchanger is a PVC integral membrane protein (Nass and Rao, 1998), required for proper sorting from the PVC to the vacuole (Bowers et al., 2000). We used a GFP tagged version of Nhx1p (kind gift of Dr. Rajini. Rao, Johns Hopkins University) that has previously been shown to complement the null Δnhx1 mutant as well have the identical localization as a version of Nhx1p that has a shorter HA tag (Nass and Rao, 1998).

Our results indicate that PVC morphology of vps mutants adds another level of characterization of the vacuolar protein sorting pathway and may be an effective method of classifying these vps mutants into functional categories.

2 Materials and methods

2.1 Yeast manipulation

Yeast deletion mutants were purchased from ResGen or Invitrogen (Carlsbad, CA). The S. cerevisiae strains were transformed using the Frozen EZ Yeast Transformation II Kit (Zymo Research, Orange, CA) following the manufacturer's instructions. The 2-micron Nhx1p plasmid (pRS425 expressing Nhx1p-GFP, kind gift of Dr. Rajini Rao, Johns Hopkins University) previously shown to complement the Δnhx1 null mutant (Nass and Rao, 1998) was used. The cells were incubated at 30°C for 45min and then spread on selective plates lacking leucine. Colonies were seen several days later after incubating at 30°C.

2.2 Microscopy

Liquid cultures of strains carrying the Nhx1p-GFP plasmid were grown overnight in selective media lacking leucine at 30°C. The following morning they were back diluted in selective media lacking leucine and allowed to grow to mid-log phase at 30°C. Living cells (1ml) were harvested by centrifugation, washed once with sterile Millipore water, and resuspended in 20ml Fluoromount-G (Southern Biotech, Birmingham, AL), placed on microscope slides, viewed on a Zeiss Axioscope or viewed on an Olympus BX-51 and imaged using a Spot RT digital camera (Diagnostic Instruments, Sterling Heights, MI). Living cells were imaged using both FITC filters and differential interference contrast (DIC) (also known as Nomarski) optics.

3 Results

In order to understand better the protein functions of some of the VPS proteins, PVC structure was probed in live yeast cells with varying vps deletion genotypes (Table 1). We began with an analysis of several vps mutants whose gene products have been studied and characterized. Understanding the PVC structure in cell strains in which the defects in the VPS pathway were known would allow for a more thorough analysis of deletion mutants whose protein deficiencies are not understood.


Table 1.

Yeast strains used in this study

Strain numberGenotypeSource
BY4742MATa his3Δ1 leu2D0 lys2Δ0 ura3Δ0Yeast deletion project
10975BY4742 vps29two colonsKanMXYeast deletion project
11271BY4742 vps35two colonsKanMXYeast deletion project
11370BY4742 vps26two colonsKanMXYeast deletion project
11845BY4742 vps5two colonsKanMXYeast deletion project
12132BY4742 vps30two colonsKanMXYeast deletion project
12388BY4742 vps17two colonsKanMXYeast deletion project
13966BY4742 vps54two colonsKanMXYeast deletion project
14070BY4742 vps61two colonsKanMXYeast deletion project
14208BY4742 vps74two colonsKanMXYeast deletion project
14318BY4742 vps52two colonsKanMXYeast deletion project
14319BY4742 vps72two colonsKanMXYeast deletion project
14471BY4742 vps73two colonsKanMXYeast deletion project
14771BY4742 vps62two colonsKanMXYeast deletion project
15091BY4742 vps51two colonsKanMXYeast deletion project
15170BY4742 vps63two colonsKanMXYeast deletion project
15231BY4742 vps65two colonsKanMXYeast deletion project
15269BY4742 vps38two colonsKanMXYeast deletion project
15554BY4742 vps66two colonsKanMXYeast deletion project
16727BY4742 vps71two colonsKanMXYeast deletion project
16797BY4742 vps53two colonsKanMXYeast deletion project
16842BY4742 vps55two colonsKanMXYeast deletion project
16929BY4742 vps70two colonsKanMXYeast deletion project


3.1 Analysis of wild type cells

As previously reported, Nhx1p is an Na+/H+ transporter that resides in the PVC (Nass and Rao, 1998). Using a GFP-Nhx1p construct the structure and morphology of the PVC were probed. Wild type cells contain between 1 and 3 PVC structures; these organelles are juxtaposed next to the vacuole (Nass and Rao, 1998) and as seen in Fig. 1. Thus, wild type PVC morphology consists of several membrane-bound components per cell, each of which is situated next to the vacuole.


Fig. 1

PVC structure in wild type cells. Living cells (strain #4742) expressing Nhx1p-GFP were mounted in Fluoromount-G and imaged using differential interference contrast (top) and FITC optics (bottom).


3.2 Analysis of cells lacking one of the retromer components

The retromer, a multiprotein complex, has been implicated in the retrieval and recycling of receptors from the PVC to the Golgi (Seaman et al., 1998). All of the subunits of the retromer have been copurified and found to associate with PVC to Golgi recycling vesicles. Interestingly, two of the retromer components are of the VPS Class A: Vps29p and Vps35p; while two are of the VPS Class B proteins, Vps5p and Vps17p; and one is a Class F protein, Vps26p (Seaman et al., 1998). In order to determine if PVC structure might be a better determinant of protein function than VPS Class identity, we looked at the PVC structure of single deletion mutants of all of the subunits of the retromer (Fig. 2A–E). Indeed we found that the PVC of each of the strains lacking one component of the retromer showed diffuse punctate staining throughout the cytosol (Fig. 2A–E). These punctate structures were generally smaller in size and always more numerous than the PVC compartment seen in wild type cells (compare to Fig. 1).


Fig. 2

PVC structure in cells lacking one component of the retromer. Living cells expressing Nhx1p-GFP were mounted in Fluoromount-G and imaged using differential interference contrast (top) and FITC optics (bottom). (A) Strain #11845 Δvps5, (B) strain #12388 Δvps17, (C) strain #11370 Δvps26, (D) Strain #10975 Δvps29, and (E) strain #11271 Δvps35.


3.3 Analysis of cells lacking one of the GARP/VFT components

Another multiprotein complex, termed the GARP (Golgi associated retrograde protein) complex (Conibear and Stevens, 2000) or VFT (Vps 53 tethering) complex (Siniossoglou and Pelham, 2001), also contains several VPS proteins. These proteins function together to mediate retrograde transport from the early endosome and the PVC to the Golgi. Vps52p/Vps53p/Vps54p functions at the vesicle to facilitate docking to the Golgi (Conibear and Stevens, 2000) and Vps51p has been implicated in regulation of the Vps52p/Vps53p/Vps54p complex and linking of this complex to the Golgi t-SNARE Tlg1p (Conibear et al., 2003; Reggiori et al., 2003; Siniossoglou and Pelham, 2002). We find that cells lacking either VPS51, VPS52, VPS53, or VPS54 all have a roughly wild type PVC appearance (Fig. 3A–D).


Fig. 3

PVC structure in cells lacking one component of the GARP/VFT complex. Living cells expressing Nhx1p-GFP were mounted in Fluoromount-G and imaged using differential interference contrast (top) and FITC optics (bottom). (A) Strain #15091 Δvps51, (B) strain #14318 Δvps52, (C) strain #16797 Δvps53, and (D) strain #13966 Δvps54.


3.4 Analysis of cells lacking one Class A VPS gene

When deleted, Class A genes show a wild type vacuolar morphology (Banta et al., 1988). However, these VPS gene products work at disparate locations in the vacuolar protein sorting pathway. We studied the PVC morphology of several strains in which one Class A gene was mutated. We looked at the Class A mutants functioning in the retromer complex, vps29 and vps35 (Fig. 2D and E, respectively), and several that do not function in the retromer complex. In strains deleted for VPS55, we saw that the PVC appeared as in wild type cells (Fig. 4A). Vps55p functions to traffic proteins from the PVC to the vacuole; additionally strains lacking VPS55 have a defect in endocytosis (Belgareh-Touzé et al., 2002). Vps38p has also been assigned to the Class A mutant category. Vps38p has been implicated in regulating the phosphatidyl inositol 3 (PI3)-kinase Vps34p in both forward transport from the Golgi to the PVC (Kihara et al., 2001) and recycling events from the PVC to the Golgi (Burda et al., 2002). Cells lacking Vps38p have a wild type PVC morphology (Fig. 4B). Another Class A protein, Vps30p, functions in autophagy (Kametaka et al., 1998) as well as being implicated in both forward (Kihara et al., 2001) and recycling events (Seaman et al., 1997). We found that in cells lacking VPS30, the PVC looked like wild type cells (Fig. 4C). Thus, several of the Class A VPS mutants have disparate sites of action as well as PVC morphology.


Fig. 4

PVC structure in cells lacking one Class A VPS gene. Living cells expressing Nhx1p-GFP were mounted in Fluoromount-G and imaged using differential interference contrast (top) and FITC optics (bottom). (A) Strain #16842 Δvps55, (B) strain #15269 Δvps38, and (C) strain #12132 Δvps30.


3.5 Analysis of uncharacterized mutants

Many vps mutants have been identified since the initial screen and selection for mutants lacking the ability to correctly localize CPY (Avaro et al., 2002; Bonangelino et al., 2002; Entian et al., 1999). Many of the gene products of these vps mutants have not yet been assigned a molecular function. We chose a subset of these uncharacterized vps mutants to begin to analyze their PVC morphology, hoping to reveal a potential cellular function of the gene products, based upon our analysis of PVC structure. We saw that in the majority of the mutant strains lacking one VPS gene, the PVC appeared as in wild type cells (vps62, vps63, vps66, vps70, vps71, vps72, vps73, and vps74) (Fig. 5). VPS63, VPS70, and VPS74 have been classified as Class A VPS mutants; VPS66, VPS71, VPS72, and VPS73 have been classified as Class B mutants; finally, VPS62 has been identified as Class F (Bonangelino et al., 2002; Bowers and Stevens, 2005). In strains lacking VPS61 or VPS65, many small punctate GFP fluorescing structures were seen (Fig. 5), reminiscent of cells lacking a component of the retromer. VPS61 has been categorized as Class B and VPS65 as Class F (Bowers and Stevens, 2005). Hopefully our further characterization of these mutants as well as others will help elucidate the function of these genes' products.


Fig. 5

PVC structure in uncharacterized vps mutants. Living cells expressing Nhx1p-GFP were mounted in Fluoromount-G and imaged using differential interference contrast (top) and FITC optics (bottom). (A) Strain #14070 Δvps61, (B) strain #14771 Δvps62, (C) strain #15170 Δvps63, (D) strain #15231 Δvps65, (E) strain #15554 Δvps66, (F) strain #16929 Δvps70, (G) strain #16727 Δvps71, (H) strain #14319 Δvps72, (I) strain #14471 Δvps73, and (J) strain #14208 Δvps74.


4 Discussion

We have begun to unravel interactions between VPS gene products using PVC structure as a probe for protein function. We have shown that mutants of the same vacuole morphology class have distinctly different PVC structure. This PVC structure is more consistent with the published function or site of action of each protein than with the vacuolar morphology class.

For instance, all components of the retromer, regardless of vacuolar morphology class, have the same PVC morphology (Fig. 2). The retromer is a pentameric protein complex composed of Vps5p, Vps17p, Vps26p, Vps29p, and Vps35p. This complex associates with recycling vesicles from the PVC back to the Golgi and is involved in cargo selection and formation of the vesicles themselves (Seaman, 2005). Strikingly, although all of these proteins compose the retromer and can be crosslinked and immunoprecipitated together (Seaman et al., 1998), the components of the retromer comprise three of the six VPS Classes. VPS29 and VPS35 are of the Class A; VPS26 is in Class F; and VPS5 and VPS17 are in Class B (Seaman et al., 1998). Our PVC morphology of deletion strains in each of these genes demonstrates that the PVC in each is composed of small, numerous, punctate structures. Thus, the phenotypes of PVC structure and localization better indicate that these gene products are working together than the original VPS classification.

The GARP/VPT complex (Vps51p/Vps52p/Vps53p/Vps54p) is required for retrograde transport from the PVC and the early endosomes, and cell strains lacking any of the components have a distinct tubulo-vacuolar morphology (Conibear et al., 2003; Conibear and Stevens, 2000). The gene products of VPS52, VPS53, and VPS54 form a complex that is required for recycling from the PVC to the Golgi (Conibear and Stevens, 2000) and Vps51p tethers this complex to Tlg1p (Conibear et al., 2003; Reggiori et al., 2003; Siniossoglou and Pelham, 2002). Deletion of VPS52, VPS53, and/or VPS54 gives an identical phenotype of a blockage of recycling from the PVC to the Golgi, including missorting and secreting 70% of newly synthesized CPY, degradation of Vps10p with a half-life of 2h; while deletion of VPS51 shows a less severe phenotype of only a 40% missorting of CPY and a degradation of Vps10p with a half-life of 3h (Conibear et al., 2003). These phenotypes show that strains with a deletion of VPS52/VPS53/VPS54 or of VPS51 have qualitatively the same phenotype but with variations. Moreover, deletion of any of the three genes VPS52/VPS53/VPS54 results in a rapid degradation of the other two components of the complex (Conibear and Stevens, 2000), while deletion of VPS51 does not result in degradation of Vps52p, Vps53p, or Vps54p (Conibear et al., 2003). Thus, Vps51p was identified later as a component of the GARP/VFT complex and has less severe deletion phenotype, leading the authors to hypothesize that Vps51p is a regulatory component of the GARP/VFT complex (Conibear et al., 2003). Our PVC morphology data support that Vps51p is indeed a member of the originally identified complex. Cell strains lacking one of the components of the GARP/VPT complex all have the same PVC morphology (Fig. 3). Thus, our PVC morphology data are consistent with the interactions of oligomeric complex of Vps51p/Vps52p/Vps53p/Vps54p.

In contrast, mutant strains that have been classified into the Class A vacuolar morphology have not been placed into the same functional localization in the cell. Consistent with this we have found that Class A mutants have distinct PVC morphology, depending on where the gene product deleted in the mutant functions within the cell. Vps30p and Vps38p have been identified as working together in a regulatory complex that associates with Vps34p to modulate levels of PI3 in the cell to regulate CPY sorting to the vacuole (Kihara et al., 2001). We found that the PVC structure of cell strains mutated for either of these genes has the phenotype of a wild type appearing PVC (Fig. 4B and C); thus wild type PVC phenotype in these two cell strains is consistent with their functioning together in the cell. We showed that the Class A mutants that are in the retromer complex have the same PVC phenotype as the other members of the retromer, even though they are of disparate VPS Classes (Fig. 2). Vps55p is involved in protein sorting from the PVC to the vacuole, although its molecular function remains obscure (Belgareh-Touzé et al., 2002). We have shown that the PVC in cells lacking VPS55 is also wild type looking (Fig. 4A). Vps55p has not yet been identified to work with any other proteins, so we cannot test whether its interaction partners have the same PVC phenotype.

We have also shown that the most of the vps mutants to which a protein function has not yet been ascribed have a wild type appearing PVC structure (Fig. 5). These include Class A mutants, VPS63, VPS70, and VPS74; Class B mutants VPS66, VPS71, VPS72, and VPS73; and the Class F mutant VPS62. We did see two unclassified vps mutants that had a phenotype of small numerous punctate GFP structures, vps61 and vps65, similar to the phenotype seen in cell strains with loss of a component of the retromer. However, VPS61, VPS63, and VPS65 are dubious open reading frames (Bowers and Stevens, 2005), so these data on the function of these VPS gene products may be spurious.

Interestingly, there is contradictory information about Vps74p that our studies may help to resolve: Vps74p has been shown to be associated by yeast two hybrid with Vps26p, a component of the retromer (Ito et al., 2001); however, the mammalian homologue of VPS74, GMx33, is a trans-Golgi matrix protein involved in sorting from the Golgi (Snyder et al., 2006). Our data seem to indicate that Vps74p is not a member of the retromer, although it may interact with Vps26p by virtue of its potential localization, gleaned from the mammalian studies.

We demonstrate in this work that PVC morphology may be a better indicator of a VPS protein's function or site of action that the original vacuolar protein sorting mutant class system (Banta et al., 1988, and Kucharczyk and Rytka, 2001). We have begun to use this PVC morphology classification system to try to determine the cellular function of some of the unclassified vps mutants. We are continuing to classify vps mutants using PVC structure hoping to be able to elucidate the function of several as of yet unstudied VPS proteins.

Acknowledgements

P.A.M. was funded by an American Heart Association New York State Affiliate Scientist Development Grant (#0130472T), J.M.H. was funded by a grant from the Holmberg Association (Jamestown, NY), and M.D.E. was funded by the Salt River Project Life Sciences Scholarship (Phoenix, AZ).

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Received 4 January 2007/16 April 2007; accepted 17 April 2007

doi:10.1016/j.cellbi.2007.04.008


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)