Studies combining electron microscopy morphometry, pulse-chase analysis of peroxisomal protein trafficking in vivo, and the isolation and protein characterization of distinct peroxisomal subforms have shown that yeast peroxisomes do not grow and divide at the same time (Veenhuis and Goodman, 1990
; Tan et al., 1995
; Titorenko et al., 2000
). There appears to be at least two different temporal patterns of peroxisome growth and division. In the yeast Candida boidinii
(Veenhuis and Goodman, 1990
), an initial event in peroxisome development is the extensive proliferation of immature peroxisomal vesicles containing only minor amounts of matrix proteins. This large increase in the number of immature peroxisomes by division precedes their growth through the import of membrane and matrix proteins and their conversion to mature organelles containing the complete set of peroxisomal proteins (Veenhuis and Goodman, 1990
). The timing of events of peroxisome growth and division is different in Y. lipolytica
. In this organism, the growth of immature peroxisomal vesicles, which is accomplished by the import of matrix proteins, and their development into mature peroxisomes occur before completely assembled mature peroxisomes undergo division (Titorenko et al., 2000
). Similar temporal patterns of peroxisome growth and division have been observed for the yeast Hansenula polymorpha
(Tan et al., 1995
). In human cells, both immature peroxisomal vesicles and mature peroxisomes are proposed to be able to divide (Gould and Valle, 2000
). However, the division of immature peroxisomes before their growth and maturation by peroxisomal protein import can be seen only in some peroxin-deficient fibroblasts following reactivation or reexpression of an originally defective peroxin-encoding gene (Matsuzono et al., 1999
; South and Gould, 1999
; Sacksteder and Gould, 2000
). In normal human cells, in contrast, conversion of immature peroxisomal vesicles to mature peroxisomes by membrane and matrix protein import may occur before peroxisomes undergo division (Gould and Valle, 2000
Members of the Pex11 family of peroxins, including Pex25p (Smith et al., 2002
) and Pex27p (Tam et al., 2003
; Rottensteiner et al., 2003b
) of S. cerevisiae
, have been shown to effect peroxisome division in different organisms (Erdmann and Blobel, 1995
; Marshall et al., 1995
; Sakai et al., 1995
; Li and Gould, 2002
; Li et al., 2002
). The dynamin-like protein Vps1p has also been implicated in this process (Hoepfner et al., 2001
), and we recently showed that the peroxisomal integral membrane proteins Pex28p and Pex29p are also involved in controlling the number, size, and separation of peroxisomes in S. cerevisiae
(Vizeacoumar et al., 2003
). Here, we have identified three novel peroxisomal proteins encoded by the ORFs YLR324w, YGR004w,
of S. cerevisiae
and demonstrated that these proteins also act to control peroxisome size and number in this organism.
The identification of novel proteins required for peroxisome biogenesis in S. cerevisiae
through their sequence similarity with known peroxins in other organisms enabled the identification of Pex28p and Pex29p (Vizeacoumar et al., 2003
Pex23p is a peroxisomal integral membrane protein required for peroxisome assembly in Y. lipolytica
that shares extensive sequence similarity to three proteins of unknown function and unknown localization encoded by the ORFs YLR324w, YGR004w,
of the S. cerevisiae
genome. Genomically encoded protein A chimeras of Ylr324p, Ygr004p, and Ybr168p were shown by a combination of confocal microscopy and subcellular fractionation to be peroxisomal proteins. In their response to extraction by different chaotropic agents, Ylr324p, Ygr004p, and Ybr168p act primarily as integral membrane proteins.
Ylr324p, Ygr004p, and Ybr168p are not required for peroxisome assembly per se, as cells harboring deletions for one, two, or all three of these genes still contain peroxisomes that are unaffected in their capacity to import PTS1- or PTS2-containing proteins. These peroxisomes are functional, at least to a degree, because the cells harboring deletions of these genes are able to grow in oleic acid-containing medium with essentially the same kinetics as wild-type cells (unpublished data). YLR324w, YGR004w, and YBR168w are also apparently not required for peroxisome inheritance, because all cells deleted for one or more of these genes still contained peroxisomes after numerous cell divisions. Also, if YLR324w, YGR004w, and YBR168w had a direct role in the inheritance of peroxisomes, one might expect that a loss of peroxisomes from cells over time resulting from the impaired segregation of peroxisomes into daughter cells would lead to inhibited growth in oleic acid-containing medium for the deletion strains as compared with the wild-type strain, which was not observed.
Peroxisomes in cells deleted for the YLR324w, YGR004w, and YBR168w genes are not normal and show distinctive phenotypic differences from wild-type peroxisomes. ylr324Δ cells showed increased numbers of peroxisomes versus wild-type cells, whereas ygr004Δ and ybr168Δ cells contained not only greater numbers of peroxisomes but also enlarged peroxisomes (). Cells deleted for two of the YLR324w, YGR004w, and YBR168w genes contained increased numbers of generally enlarged peroxisomes. Cells of the strain deleted for all three genes contained increased numbers of smaller to normally sized peroxisomes. Morphometric analyses and quantification revealed a fivefold increase in the numbers of peroxisomes on average per cell for the triple deletion strain than for the wild-type strain. Although an occasional enlarged peroxisome was evident in cells deleted for all three genes, the peroxisomal phenotype of these cells strongly resembled that of cells deleted for only the YLR324w gene. The characteristics of peroxisomes of cells of the deletion strains are consistent with a role for YLR324w, YGR004w, and YBR168w in the control of peroxisome size and number within S. cerevisiae cells. Our results suggest that Ylr324p acts primarily acts as a negative regulator of peroxisome number, whereas Ygr004p and particularly Ybr168p act as negative regulators of peroxisome size. Nevertheless, Ygr004p shares some redundancy of function with Ylr324p and Ybr168p, but not vice versa, as overexpression of YGR004w in cells deleted for one or both of the YLR324w and YBR168w genes results essentially in the reestablishment of the wild-type peroxisome phenotype, but in contrast to wild-type cells, there is some evidence of peroxisome clustering. The reason why peroxisomes cluster in these overexpressing cells is unknown.
It is interesting to note that Y. lipolytica
cells deleted for the PEX23
gene also show evidence of abnormal peroxisomal divisional control. These cells lack mature peroxisomes but do accumulate small vesicular structures that contain both peroxisomal matrix and membrane proteins (Brown et al., 2000
). However, these membrane structures do not function as peroxisomes, because pex23
Δ cells cannot grow on medium containing oleic acid as the sole carbon source. Therefore, although Yl
Pex23p, like Ylr324p, Ygr004p, and Ybr168p, likely has a role in the regulation of peroxisome division, Yl
Pex23p probably does not function identically to Ylr324p, Ygr004p, or Ybr168p in this process.
Pex28p and Pex29p have been implicated in the control of peroxisome size and number (Vizeacoumar et al., 2003
). A limited yeast two-hybrid screen revealed physical interactions among Ylr324p, Ygr004p, Ybr168p, Pex28p, and Pex29p. No interactions were detected between these five proteins and Pex11p, Pex25p, and Vps1p, which also have been shown to play a role in the control of peroxisome size and division. Further experimentation is required to determine whether these interactions are direct or bridged by other proteins. It is interesting to note that Ylr324p was shown to interact with Pex29p. Some amount of Ylr324p () and of Pex29p (Vizeacoumar et al., 2003
) was always present in the 20KgS fraction in differential fractionation and in the less dense fractions during the gradient isolation of peroxisomes. Whether some portion of these proteins forms a complex and is localized to some compartment other than peroxisomes remains to be determined.
How might Ylr324p, Ygr004p, Ybr168p, Pex28p, and Pex29p act to control the abundance, size, and distribution of peroxisomes in the S. cerevisiae
cell? Cells systematically deleted for one of the YLR324w, YGR004w,
genes and one of the PEX28
genes exhibited clusters of peroxisomes typically observed in cells deleted for the PEX28
gene (Vizeacoumar et al., 2003
). Our data suggest that Pex28p and Pex29p act upstream of Ylr324p, Ygr004p, and Ybr168p in controlling peroxisome abundance and size.
Organelles are highly dynamic structures that undergo fission and fusion to control their numbers and modify their morphology in response to intracellular and extracellular cues and to permit their correct segregation at cell division. As a consequence, the maintenance of compartmental integrity by the eukaryotic cell requires the tight coordination of mechanisms controlling these events. Many proteins, including those encoded by the genes YLR324w, YGR004w, and YBR168w of S. cerevisiae, are involved in controlling peroxisome number and size in the cell. Because of their role in the control of peroxisome size and number, we propose that YLR324w, YGR004w, and YBR168w be designated as PEX30, PEX31, and PEX32, respectively, and their encoded peroxins as Pex30p, Pex31p, and Pex32p. The challenge remains to understand how the increasing number of proteins shown to be involved in controlling peroxisome number and size interplay among themselves and signal to the cell how to control its peroxisome dynamics.