Article |
Address correspondence to Richard A. Rachubinski, Department of Cell Biology, University of Alberta, Medical Sciences Building 5-14, Edmonton, Alberta T6G 2H7, Canada. Tel: (780) 492-9868. Fax: (780) 492-9278. E-mail: rick.rachubinski{at}ualberta.ca
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Abstract |
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Key Words: biogenesis; peroxin; protein similarity; open reading frame; membrane fission
* Abbreviations used in this paper: DsRed, Discosoma sp. red fluorescent protein; PBD, peroxisome biogenesis disorder; PNS, postnuclear supernatant; PTS1, peroxisome-targeting signal 1; SM, synthetic minimal.
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Introduction |
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Proteins destined for the peroxisomal matrix are synthesized on free polyribosomes and are imported posttranslationally (Lazarow and Fujiki, 1985; Subramani 1993, 1998; Subramani et al., 2000; Purdue and Lazarow, 2001). Most peroxisomal matrix proteins are sorted by tripeptide peroxisome-targeting signal 1 (PTS1)* located at their carboxy termini (Gould et al., 1987, 1989, 1990; Aitchison et al., 1991; Swinkels et al., 1992), whereas a limited subset of proteins are sorted by a nonapeptide PTS2 at their amino termini (Osumi et al., 1991; Swinkels et al., 1991; Glover et al., 1994b; Waterham et al., 1994). A few exceptional matrix proteins are targeted by internal PTSs, which remain largely uncharacterized (Small et al., 1988; Purdue et al., 1990; Kragler et al., 1993; Elgersma et al., 1995).
Pex5p and Pex7p are receptors specific for PTS1- and PTS2-containing proteins, respectively. These receptors are mobile, interacting with their cargo in the cytosol and docking at the peroxisomal membrane through interaction with Pex13p and Pex14p (for reviews see Subramani, 1998; Hettema et al., 1999; Terlecky and Fransen, 2000; Purdue and Lazarow, 2001; Titorenko and Rachubinski, 2001). Strikingly, the PTS1 receptor has been shown to enter the peroxisomal matrix together with its cargo and to recycle back to the cytosol after dissociation from its cargo (Dammai and Subramani, 2001). The recycled PTS1 receptor is then available for another round of peroxisomal matrix protein import. The pathway of targeting of peroxisomal membrane proteins remains relatively uncharacterized, although it appears to be independent of the pathway of matrix protein targeting. Stretches of basic amino residues on the matrix side of membrane proteins, close to a transmembrane sequence, have been proposed to act in targeting proteins to the peroxisomal membrane (McCammon et al., 1994; Dyer et al., 1996; Elgersma et al., 1997; Pause et al., 2000).
A remarkable feature of peroxisomal protein import is that folded and assembled multimeric proteins can enter the organelle (Walton et al., 1992, 1995; Glover et al., 1994a; McNew and Goodman, 1994). The oligomerization of some of these multimeric proteins is facilitated by specific chaperone molecules, as in the case of the soluble peroxisomal matrix protein thiolase and its chaperone Pex20p (Titorenko et al., 1998), or is self-assisted, as in the case of the heteropentameric fatty acylCoA oxidase complex of the yeast Yarrowia lipolytica (Titorenko et al., 2002).
Failure to assemble functional peroxisomes leads to a class of genetic diseases known as the peroxisome biogenesis disorders (PBDs), the archetype of which is Zellweger syndrome (Lazarow and Moser, 1994; Brosius and Gärtner, 2002). Defining the molecular basis of the PBDs has been an area of intense research in recent years, particularly in regards to the identification of the genes controlling peroxisome assembly, the so called PEX genes. Of the 25 PEX genes identified so far, mutations in 11 of the 13 human orthologues have been shown to cause PBDs (for reviews see Fujiki, 2000; Gould and Valle, 2000; Subramani et al., 2000; Brosius and Gärtner, 2002). The identification of additional genes involved in peroxisome assembly and elucidation of the roles of the proteins they encode would provide greater understanding of the molecular basis of these lethal disorders. The completion of the Saccharomyces cerevisiae genome sequencing project has increased the utility of this model organism for the identification of novel genes involved in peroxisome assembly. Microarray transcriptional profiling of S. cerevisiae under conditions of peroxisome induction has already led to the identification of a novel PEX gene, PEX25 (Smith et al., 2002), and knowledge of the entire coding capacity of the S. cerevisiae genome has facilitated the identification of new proteins potentially involved in peroxisome assembly by their similarity with other proteins already shown to be involved in peroxisome assembly in other organisms.
We recently reported the isolation and characterization of YlPex24p, a 61-kD integral membrane protein of peroxisomes required for peroxisome assembly in Y. lipolytica (Tam and Rachubinski, 2002). A search of protein databases revealed that YlPex24p shares extensive sequence similarity with proteins encoded by the ORFs YHR150w and YDR479c of the S. cerevisiae genome. Here we report that both YHR150w and YDR479c code for peroxisomal integral membrane proteins, and we provide evidence for their role in peroxisomal dynamics in S. cerevisiae.
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Results |
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Cells deleted for either or both of the YHR150w and YDR479c genes contain increased numbers of smaller peroxisomes that exhibit clustering
We next investigated the ultrastructure of cells incubated in oleic acidcontaining YPBO medium by transmission EM. Wild-type cells (Fig. 5 A) consistently showed individual peroxisomes well separated from one another. In contrast, cells of the yhr150 (Fig. 5 B), ydr479
(Fig. 5 C), and, particularly, yhr150
/ydr479
(Fig. 5 D) strains contained peroxisomes that exhibited clustering. 28.0, 19.2, and 20.4% of peroxisomes of cells of the yhr150
/ydr479
, ydr479
, and yhr150
strains, respectively, showed clustering, in contrast to 4.0% of peroxisomes of wild-type cells (a cluster of peroxisomes was operationally defined as three or more adherent peroxisomes). The clustered peroxisomes often showed evidence of membrane thickening between adjacent peroxisomes in the cluster. Morphometric analysis showed that cells of the deletion strains contained a greater number of peroxisomes than wild-type cells and that, on average, these peroxisomes were smaller in size than those of wild-type cells (Table I). Cells of the deletion strains contained much greater numbers of peroxisomes with areas of 0.02 µm2 or less than wild-type cells (Fig. 5 E). Nycodenz density gradient centrifugation analysis showed that peroxisomes purified from yhr150
/ydr479
cells have a greatly reduced density (peak fraction 8, 1.19 g/cm3) compared with peroxisomes from wild-type cells (peak fraction 1, 1.22 g/cm3) (Fig. 6 A). Peroxisomes isolated from cells deleted for YHR150w or YDR479c were also less dense than wild-type peroxisomes, although the differences in density were less than that observed between peroxisomes from yhr150
/ydr479
cells and wild-type peroxisomes (unpublished data). EM analysis showed that the peroxisomes purified from yhr150
/ydr479
cells still exhibited clustering and evidence of thickened peroxisomal membranes, whereas peroxisomes purified from wild-type cells were largely well separated from one another, with no evidence of membrane thickening (Fig. 6 B).
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Discussion |
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The proteins encoded by the YHR150w and YDR479c genes are not required for peroxisome assembly, as cells harboring deletions for one or both of these genes still contain peroxisomes. These peroxisomes were functional, at least to some degree, as the cells containing one or both of the gene deletions were able to grow in oleic acidcontaining medium with essentially the same kinetics as the wild-type strain (unpublished data). However, the peroxisomes in the deleted strain are not normal and show phenotypic characteristics distinct from those of wild-type peroxisomes. The peroxisomes of cells deleted for one or both of the YHR150w and YDR479c genes are more abundant, smaller, and show extensive clustering, as compared with wild-type peroxisomes. In addition, the membranes of the clustered peroxisomes of the gene deletion strains are often thickened in appearance. These characteristics of peroxisomes of the deletion strains are consistent with a role for YHR150w and YDR479c in the control of peroxisome size, number, and distribution within cells. However, it does not appear that YHR150w and YDR479c are required for peroxisome inheritance per se, as all cells deleted for one or both of these genes still contained peroxisomes after numerous cell divisions. Also, if YHR150w or YDR479c had a direct role in the inheritance of peroxisomes, one might expect that a loss of peroxisomes from cells over time resulting from impaired segregation of peroxisomes into daughter cells would lead to decreased kinetics of growth in oleic acidcontaining medium for the deletion strains when compared to the wild-type strain, which, as reported above, was not observed. It is interesting to note that Y. lipolytica cells deleted for the PEX24 gene also show evidence of abnormal peroxisomal divisional control. These cells lack mature peroxisomes but do accumulate membrane structures that contain both peroxisomal matrix and membrane proteins (Tam and Rachubinski, 2002). However, these membrane structures are not functional peroxisomes in Y. lipolytica, as pex24 cells cannot grow on medium containing oleic acid as the sole carbon source. Therefore, although YlPex24p, like Yhr150p and Ydr479p, most likely has a role in the regulation of peroxisome division, YlPex24p probably does not function identically to Yhr150p or Ydr479p, or is modulated in its actions differently than Yhr150p and Ydr479p.
The size, number, and distribution of peroxisomes are tightly controlled by the cell. Loss of the enzymatic activities of individual peroxisomal ß-oxidation enzymes has been shown to result in pronounced changes in peroxisome size and/or number (Fan et al., 1998; Chang et al., 1999; Smith et al., 2000; van Roermund et al., 2000), due primarily to the increased levels of the remaining peroxisomal ß-oxidation enzymes. The molecular mechanisms underlying this so-called metabolic control of peroxisome abundance (Chang et al., 1999) remain essentially unknown.
In contrast, members of the Pex11 family of peroxins have been implicated as effectors of peroxisome division in multiple species (Erdmann and Blobel, 1995; Marshall et al., 1995, 1996; Sakai et al., 1995; Abe and Fujiki, 1998; Lorenz et al., 1998; Passreiter et al., 1998; Schrader et al., 1998; Li and Gould, 2002). The recently reported PEX25 gene has also been implicated in the regulation of peroxisome size and number in S. cerevisiae (Smith et al., 2002), as has the dynamin-like protein Vps1p (Hoepfner et al., 2001). Like other dynamin-related proteins, Vps1p was proposed to be involved in a membrane fission event required for the regulation of peroxisome size and abundance. The thickened membranes between some peroxisomes of a peroxisome cluster seen in cells deleted for the YHR150w and/or YDR479c genes are also suggestive of a role for Yhr150p and Ydr479p in controlling fission of the peroxisomal membrane.
How might Yhr150p, Ydr479p, Pex11p, Pex25p, and Vps1p act and interact to control the abundance, size, and distribution of peroxisomes in the S. cerevisiae cell? We sought to get some insight into this question by determining the effects on peroxisome morphology of overexpressing the genes for these proteins in wild-type cells and cells deleted for the different genes.
Overexpression of the PEX11 gene in the pex11 genetic background has been reported to result in large numbers of small peroxisomes (Marshall et al., 1995). In contrast, overexpression of PEX25, VPS1, YHR150w, and YDR479c in their respective gene deletion backgrounds does not result in the production of large numbers of small peroxisomes but instead restores the wild-type peroxisomal phenotype. Considering the proliferation of peroxisomes as a two-step pathway, namely division of peroxisomes and separation of peroxisomes, overexpression of the PEX11 gene either in wild-type cells or in cells of the various deletion strains leads to a significant proliferation of peroxisomes, which remain, for the most part, adherent to one another. Thus, Pex11p plays a central and positive regulatory role in the division step of peroxisome proliferation but has little, or no readily apparent, role in the separation step of the process. The presence of reduced numbers of enlarged peroxisomes in pex25
(Smith et al., 2002; Fig. S1) and vps1
cells (Hoepfner et al., 2001; Fig. S1) suggests that Pex25p and Vps1p function, in addition to Pex11p, in the divisional step of peroxisome proliferation.
Upon completion of peroxisome division, peroxisomes must be separated from one another. Yhr150p and Ydr479p are two proteins required for this process, as their absence leads to an arrest or retardation of the peroxisome proliferation pathway, leading to the presence of clusters of peroxisomes with evidence of thickened membranes sometimes occurring between adjacent peroxisomes. Significant recovery of the wild-type peroxisomal phenotype by overexpression of PEX25 or VPS1 in cells deleted for one or both of the YHR150w or YDR479c genes implies that Pex25p and Vps1p have roles in the separation of peroxisomes in addition to their roles in peroxisome division discussed above. In contrast, overexpression of PEX11 in cells deleted for one or both of the YHR150w or YDR479c genes did not result in the reappearance of wild-type peroxisomes, and peroxisomes remained clustered and sometimes exhibited membrane thickening between adjacent peroxisomes, as in the original strains deleted for YHR150w and/or YDR479c. Therefore, Pex11p appears to function primarily or only at the divisional step of peroxisome proliferation but not at the separation step.
Organelles are highly dynamic structures that undergo fission and fusion processes to allow cells to respond to intracellular and extracellular cues and to allow for their correct segregation at cell division. The maintenance of compartmental integrity in the eukaryotic cell, therefore, requires tight control mechanisms for these events. In the control of peroxisome number, size, and distribution, our data suggest that Pex11p plays a preeminent role in controlling peroxisome division, whereas Pex25p, Vps1p, and the newly identified peroxisomal proteins Yhr150p and Ydr479p all play a prominent role in controlling the separation of peroxisomes from one another. Because of their role in peroxisome dynamics, we suggest that YHR150w and YDR479c be designated as PEX28 and PEX29, respectively, and their encoded peroxins as Pex28p and Pex29p. The challenge for the future lies in understanding further the interplay amongst these proteins and the signaling events they respond to and initiate in order to control peroxisomal dynamics in the cell.
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Materials and methods |
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Protein A tagging of candidate proteins
Genes were genomically tagged with the sequence encoding Staphylococcus aureus protein A by homologous recombination using PCR-based integrative transformation into parental BY4742 haploid cells (Aitchison et al., 1995; Dilworth et al., 2001).
Microscopy
Strains encoding protein A chimeras and transformed with the plasmid pDsRedPTS1 were grown in SM medium for 12 h and then incubated in YPBO medium for 8 h. Cells were processed for immunofluorescence microscopy as previously described (Pringle et al., 1991; Tam and Rachubinski, 2002). Protein A chimeras were detected with rabbit antiserum to mouse IgG (ICN Biomedicals) and FITC-conjugated goat antirabbit IgG. Images were captured on an LSM510 META (Carl Zeiss MicroImaging, Inc.) laser scanning microscope or with a digital fluorescence camera (Spot Diagnostic Instruments). Whole cells and subcellular fractions were processed for EM as previously described (Eitzen et al., 1997).
Morphometric analysis of peroxisomes
For each strain analyzed, electron micrographs of 50 randomly selected cells at a magnification of 17, 000 were scanned, and the areas of individual cells and of individual peroxisomes were determined by counting the number of individual pixels in a particular cell or peroxisome with Image Tool for Windows, Version 2.00 (University of Texas Health Science Center). To determine the average area of a peroxisome, the total peroxisome area was calculated and divided by the total number of peroxisomes counted. To quantify peroxisome number, the numerical density of peroxisomes (number of peroxisomes per µm3 of cell volume) was calculated by the method of Weibel and Bolender (1973) for spherical organelles as follows. First, the total number of peroxisome profiles was counted and reported as the number of peroxisomes per cell area assayed (NA). Next, the peroxisome volume density (VV) was calculated for each strain (total peroxisome area/total cell area assayed). Using the values VV and NA, the numerical density of peroxisomes was determined (Weibel and Bolender, 1973).
Subcellular fractionation and isolation of peroxisomes
Subcellular fractionation and peroxisome isolation were done essentially as previously described (Bonifacino et al., 2000; Smith et al., 2002). In brief, cells grown overnight in YPD medium were transferred to YPBO medium and incubated for 8 h. Cells were harvested, washed, and converted to spheroplasts by digestion with Zymolyase 100T (1 mg/g of cells) in 50 mM potassium phosphate, pH 7.5, 1.2 M sorbitol, 1 mM EDTA for 1 h at 30°C. Spheroplasts were lysed by homogenization in buffer H (0.6 M sorbitol, 2.5 mM MES, pH 5.5, 1 mM KCl) containing 1 mM EDTA and PINS (0.5 mM benzamidine, 2 µg leupeptin/ml, 2 µg aprotinin/ml, 1 µg pepstatin A/ml, 3 µg antipain/ml, 0.5 mg Pefabloc/ml). The homogenate was subjected to centrifugation for 10 min at 2,000 g to yield a PNS fraction. The PNS fraction was subjected to further differential centrifugation at 20,000 g for 30 min to yield supernatant (20KgS) and pellet (20KgP) fractions. The 20KgP fraction was resuspended in buffer H containing 11% Nycodenz and PINS, and a volume containing 5 mg of protein was overlaid onto either a 30-ml discontinuous gradient consisting of 17, 25, 35, and 50% (wt/vol) Nycodenz or a 30-ml continuous gradient of 3045% (wt/vol) Nycodenz, both in buffer H containing PINS. Organelles were separated by centrifugation at 100,000 g for 90 min in a VTi50 rotor (Beckman Coulter). Fractions of 2 ml were collected from the bottom of the gradient.
Extraction of peroxisomes
Peroxisomes were extracted as previously described (Fujiki et al., 1982; Nuttley et al., 1990). Essentially, organelles in the 20KgP fraction (50 µg of protein) were lysed by incubation in 10 volumes of Ti8 buffer (10 mM Tris-HCl, pH 8.0) containing 3x PINS on ice for 1 h and separated into pellet (Ti8P) and supernatant (Ti8S) fractions by centrifugation at 245,000 g for 1 h at 4°C in a TLA120.2 rotor (Beckman Coulter). The Ti8P fraction was resuspended in Ti8 buffer to a final protein concentration of 0.5 mg/ml, and a portion of the resuspended fraction was extracted with 0.1 M Na2CO3, pH 11.3, for 1 h on ice and then separated into supernatant (CO3S) and pellet (CO3P) fractions by centrifugation at 245,000 g in a TLA120.2 rotor at 4°C for 1 h. Proteins in the Ti8S, Ti8P, CO3S, and CO3P fractions were precipitated by addition of TCA, and the precipitates were washed with acetone. Proteins in equal portions of each fraction were separated by SDS-PAGE and analyzed by immunoblotting.
Construction of a haploid strain deleted for both the YDR479c and YHR150w genes
The homozygous deletion diploid strain yhr150w-HD (Giaever et al., 2002) was sporulated, and the tetrads were dissected to select for the haploid MATa strain. This strain was mated to the haploid MAT
deletion strain ydr479c
by replica plating to obtain a heterozygous diploid strain harboring deletions for both YHR150w and YDR479c. The diploid strain was sporulated, and tetrads from 10 heterozygous diploids were dissected by micromanipulation. All spores were grown in YPD medium, and DNA was extracted. Haploid strains carrying deletions in both the YDR479c and YHR150w genes were selected by PCR analysis.
Antibodies
Antibodies to the carboxy-terminal SKL tripeptide (Aitchison et al., 1992), thiolase (Eitzen et al., 1996), and Sdh2p (Dibrov et al., 1998) have been previously described. FITC-conjugated antirabbit IgG and rhodamine-conjugated antiguinea pig IgG (Jackson ImmunoResearch Laboratories) were used to detect primary antibodies in immunofluorescence microscopy. Rabbit antibodies to glucose-6-phosphate dehydrogenase (G6PDH) of S. cerevisiae were obtained from Sigma-Aldrich.
Analytical procedures
Extraction of nucleic acid from yeast lysates and manipulation of DNA were performed as previously described (Ausubel et al., 1994). Immunoblotting was performed using a wet transfer system (Ausubel et al., 1994), and antigenantibody complexes in immunoblots were detected by ECL (Amersham Biosciences). Protein concentration was determined using a commercially available kit (Bio-Rad Laboratories) and bovine serum albumin as a standard.
Online supplemental material
The online version of this manuscript (http://www.jcb.org/cgi/content/full/jcb.200210130/DC1) contains additional figures (Figs. S1S6) showing the ultrastructure of cells of the gene deletion strains pex11, pex15
, pex25
, and vps1
and of various strains overexpressing the YHR150w, YDR479c, PEX11, PEX25, and VPS1 genes. Cells were grown overnight in glucose-containing medium, transferred to oleic acidcontaining medium, and incubated in oleic acidcontaining medium for 8 h. Cells were then fixed and processed for EM.
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Acknowledgments |
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This work was supported by operating grant 39322 from the Canadian Institutes of Health Research to R.A. Rachubinski and J.D. Aitchison. R.A. Rachubinski holds the Canada Research Chair in Cell Biology and is an International Research Scholar of the Howard Hughes Medical Institute. Y.Y.C. Tam is the recipient of a studentship from the Alberta Heritage Foundation for Medical Research.
Submitted: 23 October 2002
Revised: 4 March 2003
Accepted: 10 March 2003
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