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Originally published In Press as doi:10.1074/jbc.M600365200 on March 6, 2006

J. Biol. Chem., Vol. 281, Issue 18, 12817-12823, May 5, 2006
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The Dynamin-like Protein Vps1p of the Yeast Saccharomyces cerevisiae Associates with Peroxisomes in a Pex19p-dependent Manner*

Franco J. Vizeacoumar{ddagger}1, Wanda N. Vreden{ddagger}, Monica Fagarasanu{ddagger}1, Gary A. Eitzen{ddagger}2, John D. Aitchison§, and Richard A. Rachubinski{ddagger}3

From the {ddagger}Department of Cell Biology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada and the §Institute for Systems Biology, Seattle, Washington 98103

Received for publication, January 13, 2006 , and in revised form, March 6, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Dynamins and dynamin-like proteins play important roles in organelle division. In Saccharomyces cerevisiae, the dynamin-like protein Vps1p (vacuolar protein sorting protein 1) is involved in peroxisome fission, as cells deleted for the VPS1 gene contain reduced numbers of enlarged peroxisomes. What relationship Vps1p has with peroxisomes remains unclear. Here we show that Vps1p interacts with Pex19p, a peroxin that acts as a shuttling receptor for peroxisomal membrane proteins or as a chaperone assisting the assembly/stabilization of proteins at the peroxisome membrane. Vps1p contains two putative Pex19p recognition sequences at amino acids 509-523 and 633-647. Deletion of the first (but not the second) sequence results in reduced numbers of enlarged peroxisomes in cells, as in vps1{Delta} cells. Deletion of either sequence has no effect on vacuolar morphology or vacuolar protein sorting, suggesting that the peroxisome and vacuole biogenic functions of Vps1p are separate and separable. Substitution of proline for valine at position 516 of Vps1p abrogates Pex19p binding and gives the peroxisome phenotype of vps1{Delta} cells. Microscopic analysis showed that overexpression of Pex19p or redirection of Pex19p to the nucleus does not affect the normal cellular distribution of Vps1p in the cytosol and in punctate structures that are not peroxisomes, suggesting that Pex19p does not function in targeting Vps1p to peroxisomes. Subcellular fractionation showed that a fraction of Vps1p is associated with peroxisomes and that deletion or mutation of the first Pex19p recognition sequence abrogates this association. Our results are consistent with Pex19p acting as a chaperone to stabilize the association of Vps1p with peroxisomes and not as a receptor involved in targeting Vps1p to peroxisomes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Organelle division is a dynamic process orchestrated by multicomponent protein complexes that drive the constriction and fission of organellar membranes (1, 2). Members of the Pex11p family of peroxins, which includes Pex25p (3) and Pex27p (4, 5) in the budding yeast Saccharomyces cerevisiae, have been shown to effect peroxisome division in different organisms (6-10). There is also evidence for a metabolic control of peroxisome division (11, 12), which may be mediated by signals derived from the beta-oxidation of fatty acids (13-15). We recently showed that the peroxisomal integral membrane proteins Pex28p through Pex32p are also involved in controlling peroxisome number and size in S. cerevisiae (16, 17); however, their exact roles in peroxisome division have yet to be determined.

One family of proteins implicated in organelle division are the dynamins. Dynamin and dynamin-like proteins are a highly conserved family of large GTPases involved in a variety of cellular processes, including endocytosis, intracellular protein trafficking and organelle partitioning (18, 19). In S. cerevisiae, the dynamin-like protein Dnm1p has been shown to assemble in a multicomponent complex at the outer mitochondrial membrane and mediate the division of mitochondria (20), whereas the dynamin-like protein vacuolar protein sorting protein 1 (Vps1p) has been implicated in peroxisome fission (21). Cells lacking Vps1p have fewer and enlarged peroxisomes as compared with wildtype cells. Vps1p is related to mammalian DLP1. Peroxisome elongation and constriction can occur independently of DLP1, whereas peroxisome fission requires DLP1 (22). Pex11p has been suggested to recruit DLP1 to peroxisomes, although no direct interaction between these two proteins has been observed (23). We have reported that oversynthesis of Pex11p in vps1{Delta} cells results in the formation of peroxisomes that are similar in size and number to the peroxisomes of wild-type cells, suggesting that overproduction of Pex11p can overcome the requirement for Vps1p in controlling peroxisome fission (16). Vps1p has also been suggested to regulate the actin cytoskeleton through its interaction with proteins capable of promoting actin assembly (24-28). Interestingly, a vps1{Delta} rho1{Delta} double mutant of S. cerevisiae showed accumulation of actin on peroxisomes, suggesting that actin is reorganized/disassembled before peroxisome fission (29).

All of these observations support a direct role for Vps1p in peroxisome fission at the level of the peroxisome itself, but it remains to be determined how Vps1p could associate with peroxisomes. One protein that could recruit Vps1p to peroxisomes is Pex19p. Pex19p is a farnesylated peroxin involved in peroxisome membrane biogenesis (30, 31). Studies suggest that Pex19p functions as an import receptor for peroxisomal membrane proteins (PMP)4 and/or as a chaperone acting in the assembly or stabilization of PMPs in the cytosol or at the peroxisomal membrane (32-36). Here we show that the association of Vps1p with peroxisomes is dependent on Pex19p and that the interaction of Vps1p with Pex19p is required for peroxisomal fission. Our findings are consistent with a role for Pex19p as an assembly or stabilization factor for Vps1p at the peroxisome.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Strains and Culture Conditions—The S. cerevisiae strains used in this study are listed in Table 1. Strains were cultured at 30 °C. Media components were as follows: YPD (1% yeast extract, 2% peptone, 2% glucose); YPBO (0.3% yeast extract, 0.5% peptone, 0.5% K2HPO4, 0.5% KH2PO4, 1% Brij 35, 1% oleic acid); synthetic minimal (SM) medium (0.67% yeast nitrogen base without amino acids, 2% glucose, 1x Complete Supplement Mixture (Bio 101) without histidine and/or leucine); and YNBD (0.67% yeast nitrogen base without amino acids, 2% glucose, containing histidine, leucine, and uracil, each at 30 µg/ml).


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  		TABLE 1
S. cerevisiae strains used in this study

 
Plasmids—The plasmid pDsRed-PTS1 has been described previously (3). Genes to be overexpressed were amplified by PCR and cloned into the plasmid YEp13 (37). For overexpression, the PEX19 gene included 400 bp of upstream sequence and 347 bp of downstream sequence. The plasmid pGFP/HIS5 was constructed from the plasmid pProtA/HIS5 (38) by exchange of the sequence encoding protein A (ProtA) with sequence encoding an improved version of GFP (GFP+) from Aequoria victoria (39). The plasmid pGFP/NAT (nourseothricin) was constructed from pGFP/HIS5 by exchange of the HIS5 auxotrophic marker for the gene encoding resistance to the drug nourseothricin. pNLS-PEX19 and pNLS-GFP-PEX19 were constructed in pRS316 and contained sequence encoding either the nuclear localization signal (NLS) of Mad1p NLS-Pex19p or NLS-GFP-Pex19p under the control of the PHO4 promoter.

GFP Tagging of Genes—Genes were genomically tagged with the sequence encoding GFP by homologous recombination with a PCR-based integrative transformation of parental BY4742 or pex19{Delta} haploid cells (40). The functionality of fusion proteins was confirmed by the lack of a mutant phenotype in transformed strains.

Construction of Yeast Strains Mutated in the VPS1 Gene—In-frame deletions of the regions of the VPS1 gene coding for putative Pex19p recognition sequences were constructed by overlapping PCR followed by genomic integration of the mutated genes at the VPS1 locus of the haploid parental strain BY4742 to make the strains vps1{Delta}509-523 and vps1{Delta}633-647. Codon 516 of the VPS1 gene encoding valine was changed to a codon encoding proline by site-directed mutagenesis, and the mutated gene was genomically integrated at the VPS1 locus of the strain BY4742 to make the strain vps1V516P.

Microscopy—Strains synthesizing GFP chimeric proteins or transformed with the plasmid pDsRed-PTS1 were grown in SM medium for 12 h and then incubated in YPBO medium for 8 h. Images were captured on a LSM510 META (Carl Zeiss) laser scanning microscope or on an Olympus BX50 microscope equipped with a digital fluorescence camera (Spot Diagnostic Instruments). Cells were processed for immunofluorescence microscopy (41) and electron microscopy (42).

Two-hybrid Analysis—Physical interactions between Pex19p and Vps1p or Vps1pV516P were detected using the Matchmaker two-hybrid system (BD Biosciences). Chimeric genes were made by amplifying the open reading frames for Pex19p, Vps1p, and Vps1pV516P and ligating them in-frame and downstream of the DNA encoding the transcription-activating domain (AD) and the DNA-binding domain (BD) of the GAL4 transcriptional activator in the plasmids pGAD424 and pGBT9, respectively. Cells of S. cerevisiae strain HF7c were transformed simultaneously with a pGAD424-derived plasmid and a pGBT9-derived plasmid. Transformants were grown on SM agar medium lacking tryptophan, leucine, and histidine. Growth on this medium indicates an interaction between two chimeric proteins.

Secretion of Vacuolar Carboxypeptidase Y—Detection of the secretion of vacuolar carboxypeptidase Y (CPY) was performed as described previously (43), with minor modifications. Briefly, cells were grown in YPD medium to an A600 of 0.5 and then spotted onto YPD agar plates in 2-fold serial dilutions. The cells were overlaid with nitrocellulose and then incubated at 30 °C for 24 h. The nitrocellulose was lifted from the plate, rinsed with water, and subjected to immunoblotting with anti-CPY antibody. Antigen-antibody complexes were detected by enhanced chemiluminescence (Amersham Biosciences).

Vacuolar Staining—Cells grown overnight at 30 °C in YPD medium were subcultured in YPD medium at 30 °C until they reached an A600 of 0.5. 1 µl of 8 mM FM4-64 (44) was added to 100 µl of cells, and the cells were incubated for 30 min at 30 °C. The cells were washed with water and incubated in fresh YPD medium for 90 min at 30 °C. The cells were washed twice with water and then observed by fluorescence microscopy.

Subcellular Fractionation and Isolation of Peroxisomes—Subcellular fractionation of oleic acid-incubated cells was done as described previously (3) and involved the isolation of a post-nuclear supernatant fraction and 20,000 x g supernatant (20KgS) and pellet (20KgP) fractions enriched for cytosol and for peroxisomes and mitochondria, respectively. Peroxisomes were purified from the 20KgP fraction by isopycnic density centrifugation on Nycodenz gradients (3).

Antibodies—Antibodies to the carboxyl-terminal SKL tripeptide, peroxisomal thiolase, and to the mitochondrial enzyme Sdh2p (succinate dehydrogenase 2) have been described previously (5). Antibodies to Vps1p were raised in rabbit as described previously (45). Antibodies to CPY were raised in rabbit and were a kind gift of Dr. William Wickner (Dartmouth College). Rabbit antibodies to S. cerevisiae glucose-6-phosphate dehydrogenase were from Sigma-Aldrich. Fluorescein isothiocyanate-conjugated anti-rabbit IgG and rhodamine-conjugated antiguinea pig IgG (The Jackson Laboratory) were used to detect primary antibodies in immunofluorescence microscopy.

Analytical Procedures—Extraction of nucleic acid from yeast lysates and manipulation of DNA were performed as described previously (46). Immunoblotting was performed using a wet transfer system (46), and antigen-antibody complexes in immunoblots were detected by enhanced chemiluminescence. Protein concentration was determined using a commercially available kit (Bio-Rad) and bovine serum albumin as a standard.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Pex19p Interacts with Vps1p—Vps1p belongs to the dynamin family of proteins and is required for sorting proteins to the vacuolar compartment. Vps1p also has a role in peroxisome biogenesis, as cells deleted for the VPS1 gene contain reduced numbers of enlarged peroxisomes (21). Vps1p has been suggested to cycle from the cytosol to the surface of the peroxisome (21). The peroxin Pex19p has been implicated in peroxisome membrane biogenesis and functions either as an import receptor for PMPs or as an assembly/disassembly factor, i.e. a chaperone, for PMPs in the cytosol or at the peroxisomal membrane (32-36). Recent work has defined a consensus sequence for recognition of a protein by Pex19p (47). Vps1p contains two stretches of amino acids that conform to this consensus sequence (Fig. 1A). Yeast two-hybrid analysis showed an interaction between Vps1p and Pex19p (Fig. 1B).


Figure 1
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  		FIGURE 1.
Vps1p interacts with Pex19p. A, putative Pex19p recognition sequences in Vps1p. The sequence at the top corresponds to the consensus sequence for Pex19p recognition (47). Two amino acid sequences in Vps1p that conform to the consensus sequence for Pex19p recognition are presented, along with their positions within the primary sequence of Vps1p. B, yeast two-hybrid analysis. Full-length genes encoding Pex11p, Pex19p, and Vps1p were fused to the GAL4 binding domain (GAL4-BD) in the vector pGBT9. The resulting plasmids were cotransformed into S. cerevisiae strain HF7C with a pGAD424-derived plasmid expressing a fusion gene between the GAL4 activation domain (GAL4-AD) and PEX19. Two independent sets of transformants were tested for evidence of protein-protein interaction as shown by growth on agar medium lacking histidine, leucine, and tryptophan. No growth was observed when the parental vector pGBT9 (—) was cotransformed with the plasmid expressing GAL4-AD-Pex19p.

 
Deletion of the First Consensus Pex19p Recognition Sequence in Vps1p Yields the vps1{Delta} Peroxisomal Phenotype—Cells carrying chromosomally integrated mutant forms of VPS1 deleted for sequences encoding the first (vps1{Delta}509-523) or second (vps1{Delta}633-647) putative Pex19p recognition site were examined by immunofluorescence microscopy to observe whether they exhibited the mutant peroxisomal phenotype of vps1{Delta} cells. vps1{Delta}509-523 cells showed reduced numbers of enlarged peroxisomes characteristic of vps1{Delta} cells, whereas vps1{Delta}633-647 cells showed a peroxisomal phenotype similar to that of wild-type cells (Fig. 2). Electron microscopy confirmed the presence of enlarged peroxisomes in vps1{Delta}509-523 cells (see Fig. 5B), which were similar in size to peroxisomes of vps1{Delta} cells (see Fig. 5C) and much larger than those of wildtype cells (see Fig. 5A). Our findings show that the consensus Pex19p recognition sequence in Vps1p at amino acids 509-523 is required for Vps1p function in controlling peroxisome size and number.


Figure 2
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  		FIGURE 2.
Cells deleted for the first putative Pex19p recognition sequence of Vps1p exhibit the enlarged peroxisomal phenotype of vps1{Delta} cells. Cells were grown in YPD medium for 16 h, transferred to oleic acid-containing YPBO medium, and incubated for 8 h in YPBO medium. Wild-type BY4742 cells, vps1{Delta} cells, and cells containing genomically integrated VPS1 mutations deleted for the first (vps1{Delta}509-523) or second (vps1{Delta}633-647) putative Pex19p recognition sequence were observed by immunofluorescence microscopy with antibodies to the PTS1 tripeptide Ser-Lys-Leu (SKL) or to the PTS2-containing protein thiolase. Rabbit primary antibodies (SKL) were detected with fluorescein isothiocyanate-conjugated secondary antibodies. Guinea pig primary antibodies (thiolase) were detected with rhodamine-conjugated secondary antibodies. Scale bar, 1 µm.

 


Figure 3
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  		FIGURE 3.
Vacuolar morphology and vacuolar protein sorting are unaffected in cells deleted for the first Pex19p recognition sequence of Vps1p. A, vacuolar morphology of cells of the wild-type BY4742 and vps1{Delta} strains and of strains deleted for either the first (vps1{Delta}509-523) or second (vps1{Delta}633-647) putative Pex19p recognition sequence of Vps1p. Vacuoles were stained by the dye FM4-64 and observed by fluorescence microscopy. Scale bar, 1 µm. B, CPY secretion assay. Cells of the wild-type BY4742 and vps1{Delta} strains and of strains deleted for either the first (vps1{Delta}509-523) or second (vps1{Delta}633-647) putative Pex19p recognition sequence of Vps1p were spotted onto YPD agar in 2-fold serial dilutions and overlaid with nitrocellulose. Secretion of CPY is detected by immunoblotting with anti-CPY antibody.

 
Cells Deleted for the First Pex19p Recognition Sequence of Vps1p Are Unaffected in Vacuolar Morphology and Protein Sorting—Vacuolar biogenesis requires membrane fusion and fission events and the actin-dependent transport of vacuolar membranes. Mutations of genes involved in these processes often lead to vacuoles with aberrant morphology (48) and defects in vacuolar protein sorting (49). We stained wild-type and vps1 mutant cells with the fluorescent vacuolar vital stain FM4-64 to examine their vacuolar morphology (Fig. 3A). The majority of vps1{Delta} cells exhibited a fragmented vacuolar morphology, as has been observed previously (48, 50). Both vps1{Delta}509-523 and vps1{Delta}633-647 cells exhibited wild-type vacuolar morphology.

We also analyzed the sorting of the soluble vacuolar hydrolase carboxypeptidase Y (CPY) (Fig. 3B). Defects in vacuolar protein sorting lead to secretion of CPY (49). vps1{Delta}509-523 cells did not show evidence of enhanced secretion of CPY when compared with wild-type cells. This result suggests that the peroxisome and vacuolar biogenic roles of Vps1p are separate and separable. vps1{Delta}633-647 cells showed a slightly increased level of secreted CPY but much less than that observed for vps1{Delta} cells, suggesting that these amino acids of Vps1p may have some role in vacuolar protein sorting.


Figure 4
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  		FIGURE 4.
A point mutation within the first Pex19p recognition sequence of Vps1p leads to reduced Pex19p binding and abnormal peroxisome morphology. A, yeast two-hybrid analysis shows greatly reduced interaction between Pex19p and Vps1p containing a valine to proline substitution at position 516 in the first Pex19p consensus recognition sequence of Vps1p (Vps1pV516P). B, cells expressing Vps1pV516P contain reduced numbers of enlarged peroxisomes like vps1{Delta} cells. Cells were observed by immunofluorescence microscopy with antibodies to the PTS1 tripeptide Ser-Lys-Leu (SKL) or to the PTS2-containing protein thiolase. Rabbit primary antibodies (SKL) were detected with fluorescein isothiocyanate-conjugated secondary antibodies. Guinea pig primary antibodies (thiolase) were detected with rhodamine-conjugated secondary antibodies. Scale bar, 1 µm.

 
A Point Mutation in the First Pex19p Recognition Sequence of Vps1p Disrupts the Interaction of Vps1p with Pex19p—Substitution of a proline for any amino acid within the consensus Pex19p recognition sequence has been shown to disrupt the interaction of the PMPs Pex11p and Pex13p with Pex19p (47). Introduction of proline within the Pex19p binding region likely disrupts its {alpha}-helical conformation, which is thought to promote the association of a partner protein with Pex19p. Accordingly, we substituted proline for valine 516 (V516P) within the first putative Pex19p binding site of Vps1p (Vps1pV516P). Two-hybrid analysis showed that the V516P substitution greatly reduced the interaction of Vps1p with Pex19p (Fig. 4A). Immunofluorescence microscopy (Fig. 4B) and electron microscopy (Fig. 5D) showed that cells of a strain genomically expressing Vps1pV516P contained reduced numbers of enlarged peroxisomes, similar to cells of the vps1{Delta} strain.


Figure 5
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  		FIGURE 5.
Peroxisome ultrastructure in wild-type and vps1 mutant cells. Cells of the wild-type strain BY4742 (A) and of the vps1 mutant strains vps1{Delta}509-523 (B), vps1{Delta} (C), and vps1V516P (D) were grown in YPD medium for 16 h, transferred to YPBO medium and incubated in YPBO medium for 8 h. Cells were fixed and processed for electron microscopy. P, peroxisome. Scale bar, 1 µm.

 


Figure 6
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  		FIGURE 6.
Pex19p does not recruit Vps1p to peroxisomes. A, the subcellular distribution of the chimera Vps1p-GFP was compared with that of the peroxisomal marker DsRed-PTS1 by fluorescence microscopy of BY4742 cells overexpressing the PEX19 gene in the plasmid YEp13 and incubated in oleic acid-containing YPBO medium. Vps1p-GFP does not colocalize with DsRed-PTS1. B, directed mistargeting of Pex19p to the nucleus fails to direct Vps1p-GFP to the nucleus. Attachment of the NLS of Mad1p directs a fluorescent chimera of Pex19p (NLS-GFP-Pex19p) to the nucleus. NLS-Pex19p directs a fluorescent chimera (Pex32p-GFP) of the peroxisomal integral membrane protein Pex32p to the surface of the nucleus but fails to direct Vps1p-GFP to the nucleus. Nuclei were stained with Hoescht 33342. Scale bar, 1 µm.

 
The Subcellular Distribution of Vps1p Is Unaffected by Overproduction or Directed Mistargeting of Pex19p—Vps1p in glucose-grown cells has been shown to be primarily cytosolic with some localization in the trans-Golgi network (51). Localization of Vps1p to peroxisomes has been difficult to demonstrate (21). Because we have shown that Pex19p interacts with Vps1p, we wanted to determine whether overproduction of Pex19p would alter the subcellular location of Vps1p and possibly lead to an increased, detectable amount of Vps1p associating with peroxisomes. In wild-type BY4742 cells, Vps1p-GFP exhibited both a diffuse cytosolic fluorescence and localization to punctate structures, which did not correspond to peroxisomes labeled with the fluorescent peroxisomal marker DsRed-PTS1 (Fig. 6A). Overexpression of the PEX19 gene carried on the multicopy plasmid YEp13 in BY4742 cells did not alter the subcellular distribution of Vps1p, and no preferential association of Vps1p with peroxisomes was observed (Fig. 6A).


Figure 7
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  		FIGURE 7.
Vps1p association with peroxisomes is dependent on its interaction with Pex19p. A, wild-type BY4742 cells and cells expressing the mutant Vps1p forms Vps1p{Delta}509-523 or Vps1pV516P were incubated in oleic acid-containing medium, and their homogenates were subjected to differential centrifugation to yield a post-nuclear supernatant fraction, a 20,000 x g supernatant (20KgS) fraction enriched for cytosol, and a 20,000 x g pellet (20KgP) fraction enriched for peroxisomes and mitochondria. An equal percentage of each subcellular fraction was separated by SDS-polyacrylamide gel electrophoresis and subjected to immunoblotting with antibodies to Vps1p, thiolase (peroxisomes), Sdh2p (mitochondria), or glucose-6-phosphate dehydrogenase (cytosol). B, the 20KgP fraction from oleic acid-incubated wild-type cells was subjected to isopycnic density centrifugation on a discontinuous Nycodenz gradient. The gradient was collected in 15 equal fractions from the bottom of the centrifuge tube. An equal percentage of each fraction was subjected to SDS-polyacrylamide gel electrophoresis and subjected to immunoblotting with antibodies to Vps1p, thiolase, or Sdh2p.

 
In mammalian cells, attachment of a NLS to Pex19p has been shown to redirect Pex19p to the nucleus, which in its turn redistributes a Pex19p-associating protein from peroxisomes to the nucleus (35, 52). We therefore wanted to determine whether a nuclear-targeted chimera of Pex19p, NLS-Pex19p, containing the NLS of the spindle assembly checkpoint protein Mad1p (53, 54) could redirect the fluorescent chimera Vps1p-GFP to the nucleus. NLS-GFP-Pex19p localized to the nucleus, as expected (Fig. 6B). NLS-Pex19p was capable of directing a GFP chimera of the peroxisomal membrane protein Pex32p (17) to the outer surface of the nucleus, most probably the nuclear envelope (Fig. 6B). Complete import of Pex32p-GFP into the nucleus may have failed because of steric or conformational incompatibility of the Pex32p-GFP-NLS-Pex19p complex for nuclear import. In contrast, NLS-Pex19p did not redirect Vps1p-GFP to the nucleus from the cytosol or from punctate structures (Fig. 6B). The results of our experiments on the overproduction or directed mistargeting of Pex19p do not support a role for Pex19p in recruiting Vps1p to peroxisomes.

Vps1p Associates with Peroxisomes in a Pex19p-dependent Manner—Because our microscopic analyses provided no evidence of a peroxisomal localization for a fraction of Vps1p and did not support a role for Pex19p in recruiting Vps1p to peroxisomes, we turned to subcellular fractionation for evidence of a peroxisomal association for some Vps1p and to determine what role the interaction between Vps1p and Pex19p might play in this association.

Subcellular fractionation followed by immunoblotting with specific antibodies showed that in wild-type BY4742 cells incubated in oleic acid-containing medium, most Vps1p was localized to a 20,000 x g supernatant (20KgS) fraction enriched for cytosol (Fig. 7A). However, a small but reproducible amount of Vps1p was localized to the 20,000 x g pellet (20KgP) fraction enriched for peroxisomes and mitochondria. The Vps1p present in the 20KgP fraction was not due to cytosolic contamination of the 20KgP fraction, as the cytosolic enzyme glucose-6-phosphate dehydrogenase was found exclusively in the 20KgS fraction. Isopycnic density gradient centrifugation of the 20KgP fraction from wild-type cells showed that some Vps1p was found in fractions enriched for peroxisomes, as marked by the presence of the peroxisomal matrix protein thiolase and the absence of the mitochondrial protein Sdh2p (Fig. 7B). In cells expressing Vps1p{Delta}509-523 or Vps1pV516P, no Vps1p was found localized to the 20KgP fraction (Fig. 7A). As expected, thiolase and Sdh2p remained preferentially localized to the 20KgP fraction in cells expressing Vps1p{Delta}509-523 or Vps1pV516P. These results show that some fraction of Vps1p is associated with peroxisomes and that this association is dependent on its interaction with Pex19p through its first Pex19p recognition sequence.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Organelles are highly dynamic structures that undergo fission and fusion to control their numbers, modify their morphology in response to intracellular and extracellular cues, and permit their proper segregation at cell division. Maintenance of the overall compartmental integrity of the eukaryotic cell requires the tight coordination of mechanisms controlling these events. The dynamins and dynamin-like proteins play key roles in this process.

In yeast, fission of mitochondria occurs by a multistep pathway that involves recruitment of the dynamin-like protein Dnm1p and accessory proteins to sites on mitochondrial tubules, constriction of the mitochondrial tubules at these sites, and coordinated division of the outer and inner mitochondrial membranes to generate new tubule ends (20). Another dynamin-like protein, Vps1p, is involved in regulating peroxisome fission. Deletion of the VPS1 gene leads to cells containing a few enlarged peroxisomes (21). Vps1p has been shown to have a role in regulating cytoskeletal dynamics (28), and cells mutant for both the VPS1 gene and for the RHO1 gene encoding a small GTPase have been found to accumulate actin on peroxisomes (29). How might Vps1p regulate fission at the level of the peroxisome? Because the peroxin Pex19p has been shown to function in assembly of the peroxisomal membrane, we proposed that Vps1p could associate with peroxisomes in a Pex19p-dependent manner so as to participate in peroxisome fission. Using the consensus sequence for Pex19p recognition (47), we found two putative Pex19p binding sites in Vps1p and showed by yeast two-hybrid analysis that Pex19p indeed interacts with Vps1p.

If the association of Vps1p with peroxisomes is dependent on Pex19p, then mutations in Vps1p that disrupt its interaction with Pex19p would be predicted to give rise to the peroxisomal phenotype exhibited by vps1{Delta} cells, i.e. reduced numbers of enlarged peroxisomes. Cells expressing a mutant form of Vps1p lacking the first Pex19p binding site (Vps1p{Delta}509-523), but not the second site (Vps1p{Delta}633-647), did indeed exhibit reduced numbers of enlarged peroxisomes such as vps1{Delta} cells. A single substitution mutation within the first Pex19p recognition sequence of Vps1p, V516P, greatly diminished the interaction of Pex19p with Vps1p, and cells expressing Vps1pV516P also contained fewer and enlarged peroxisomes, similar to the peroxisome phenotype of vps1{Delta} cells.

Deletion of the first Pex19p recognition site of Vps1p did not affect vacuolar protein sorting, whereas deletion of the second site led to only a modest increase in secretion of the vacuolar hydrolase CPY. It is noteworthy that deletion of the second Pex19p recognition site spans the dynamin GTPase effector domain of Vps1p (55). This domain has been proposed to form coiled-coil structures and to bind the syntaxin-related protein Vam3p in an interaction that is essential for vacuolar fusion (50). Deletion of either the first or second Pex19p recognition site of Vps1p did not lead to abnormal vacuolar morphology, unlike deletion of the entire VPS1 gene, which results in cells exhibiting a mixed vacuolar phenotype with both enlarged and fewer vacuoles (fission mutants) and many small fragmented vacuoles (fusion mutants). This mixed vacuolar phenotype has been suggested to arise from the mutual control of membrane fission and fusion by Vps1p (50). Our findings suggest that the peroxisome biogenic and vacuole biogenic functions of Vps1p are both separate and separable.

In mammalian cells, a chimeric Pex19p containing a NLS has been shown to target to the nucleus and to direct proteins with which it interacts to the nucleus (35, 52). A chimera of S. cerevisiae Pex19p and the NLS of the nuclear protein Mad1p efficiently targeted the nuclei of yeast cells and directed a GFP-tagged variant of the PMP Pex32p to the nuclear surface, presumably the nuclear envelope, but failed to direct Vps1p-GFP to the nucleus. Instead, Vps1p-GFP was localized diffusely in the cytosol or in punctate structures of unknown identity. Overexpression of Pex19p also did not lead to increased accumulation of Vps1p-GFP in peroxisomes, and Vps1p-GFP remained diffuse in the cytosol or in punctate structures that were not peroxisomes. However, subcellular fractionation showed that some Vps1p was indeed associated with peroxisomes and that deletion of the first Pex19p recognition sequence (Vps1p{Delta}509-523) or mutation of valine to proline at position 516 in the first Pex19p recognition sequence (Vps1pV516P) in Vps1p abolished this association.

Although Pex19p is predominantly cytosolic, a small amount of it is found associated with peroxisomes (30, 31). The cytosolic fraction of Pex19p has been suggested to act as a shuttling receptor for PMPs, whereas its peroxisome-associated form may function as a chaperone, assisting the assembly of multimeric complexes or the stabilization of proteins following their binding to the peroxisome membrane (32-34, 36). Recent evidence has suggested that Pex19p could be bifunctional, acting both as a cytosolic/peroxisomal PMP chaperone and as a PMP import receptor (35). In regards to Vps1p, our results are consistent with Pex19p acting as a chaperone to stabilize the association of Vps1p with peroxisomes and not as a receptor involved in targeting Vps1p to the peroxisome.

In closing, our studies have shown that the dynamin-like protein Vps1p of S. cerevisiae can be bound by Pex19p, a peroxin involved in the assembly of the peroxisomal membrane, and that the association of Vps1p with peroxisomes is dependent on Pex19p. Disruption of the interaction between Vps1p and Pex19p results in the abnormal peroxisome phenotype of reduced numbers of enlarged peroxisomes observed in vps1{Delta} cells but no change in vacuolar morphology or vacuolar protein sorting. Therefore, the peroxisome biogenic and vacuole biogenic functions of Vps1p are apparently both separate and separable.


   FOOTNOTES
 
* This work was supported by Grant MOP-15131 from the Canadian Institutes of Health Research (CIHR) (to R. A. R.) and Grant GM067228 from the National Institutes of Health (to J. D. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Recipient of a studentship from the Alberta Heritage Foundation for Medical Research (AHFMR). Back

2 A CIHR new investigator and an AHFMR scholar. Back

3 Holder of a Canada research chair in cell biology and an international research scholar of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Dept. of Cell Biology, University of Alberta, Medical Sciences Bldg. 5-14, Edmonton, Alberta T6G 2H7, Canada. Tel.: 780-492-9868; Fax: 780-492-9278; E-mail: rick.rachubinski{at}ualberta.ca.

4 The abbreviations used are: PMP, peroxisomal membrane protein; NLS, nuclear localization signal; GFP, green fluorescent protein; CPY, carboxypeptidase Y; 20KgS, 20,000 x g supernatant; 20KgP, 20,000 x g pellet. Back


   ACKNOWLEDGMENTS
 
We thank Richard Poirier for help with confocal microscopy and Honey Chan for help with electron microscopy. A plasmid containing an insert coding for the NLS of Mad1p and GFP was a gift from Dr. Richard Wozniak (University of Alberta).



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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