Completion of the S. cerevisiae
genome sequencing project proved invaluable for the identification of a novel PEX
, by transcriptome profiling of cells grown in oleic acid–containing medium versus cells grown in glucose-containing medium (Smith et al., 2002
). The completed S. cerevisiae
genome sequence has added value in that it provides the opportunity to identify novel proteins required for peroxisome biogenesis in S. cerevisiae
through sequence similarity between proteins of unknown function encoded by S. cerevisiae
and proteins already shown to be required for peroxisome biogenesis in other organisms. We recently demonstrated the requirement of the peroxisomal integral membrane protein Pex24p for peroxisome assembly in the yeast Y. lipolytica
(Tam and Rachubinski, 2002
Pex24p was shown to share extensive sequence similarity with two proteins of unknown function and unknown localization encoded by the ORFs YHR150w
of the S. cerevisiae
genome. In this manuscript, genomically encoded protein A chimeras of Yhr150p and Ydr479p were shown by a combination of microscopic and subcellular fractionation analyses to be peroxisomal proteins. In their response to extraction by different salts, Yhr150p and Ydr479p act primarily as integral membrane proteins. However, a fraction of Ydr479p consistently acts as a soluble protein. The exact nature of this soluble form of Ydr479p and its origin (is it in equilibrium with the peroxisomal membrane form of Ydr479p?) remain under investigation.
The proteins encoded by the YHR150w
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deletions were able to grow in oleic acid–containing medium with essentially the same kinetics as the wild-type strain (unpublished data).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
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
in the control of peroxisome size, number, and distribution within cells. However, it does not appear that YHR150w
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
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 acid–containing 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
Δ cells cannot grow on medium containing oleic acid as the sole carbon source. Therefore, although Yl
Pex24p, like Yhr150p and Ydr479p, most likely has a role in the regulation of peroxisome division, Yl
Pex24p 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 β-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 β-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
; 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
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
Δ genetic background has been reported to result in large numbers of small peroxisomes (Marshall et al., 1995
). In contrast, overexpression of PEX25
, 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
Δ (Smith et al., 2002
; Fig. S1) and vps1
Δ 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.