Eukaryotic cells partition their organelle populations during cell division. Here, we report that Inp1p, a protein of unknown function encoded by the S. cerevisiae genome, is required for peroxisome inheritance. Inp1p is the first peroxisomal protein directly implicated in the inheritance of peroxisomes. Inp1p is not required for peroxisome assembly per se, because cells harboring a deletion of INP1 contain readily identifiable peroxisomes by microscopic analysis and are able to import proteins targeted by either PTS1 or PTS2 (unpublished data).
Cells deleted for INP1 incubated in oleic acid medium showed a progressive decrease in the average number and increase in the average size of peroxisomes with time. However, there was heterogeneity in the peroxisome population, with some cells containing a few enlarged peroxisomes and other cells containing peroxisomes similar in size and number to peroxisomes of wild-type cells. This heterogeneity was suggestive of a defect in peroxisome partitioning. When inp1Δ cells were cultured in medium permitting peroxisome proliferation and rapid cell division, an imbalance in the partitioning of peroxisomes became readily apparent as mother cells without peroxisomes were observed. The overall proportion of mother cells without peroxisomes increased with increasing bud size. These observations, combined with the fact that overexpression of INP1 led conversely to large numbers of buds without peroxisomes and relocation of peroxisomes to the cortical regions of cells, strongly suggested a role for Inp1p in peroxisome inheritance.
The inheritance of organelles in budding yeast consists of two complimentary processes: the retention of a subset population of an organelle in the mother cell and the ordered movement of the remaining portion of the organelle population to the forming bud. The close control of both processes is crucial to the successful distribution of the organelle from mother cell to bud. A retention mechanism within the mother cell has been described for mitochondria (Yang et al., 1999
). Retained mitochondria accumulate at the tip of the mother cell distal to the site of bud emergence (the so called “retention zone”), a process that likely involves the actin cytoskeleton. Retention mechanisms also operate in the bud. In this study we showed that, similar to mitochondria, peroxisomes are actively retained in the mother cell. Both organelles and molecules have been shown to remain anchored to the bud cell cortex at discrete locations, as demonstrated for mitochondria (Simon et al., 1997
), ASH1 mRNA (Long et al., 1997
; Takizawa et al., 1997
), and the protein chitin synthase 3 (DeMarini et al., 1997
). Recently, the Rab-like protein Ypt11p was shown to be required for the retention of newly inherited mitochondria within buds of S. cerevisiae
(Boldogh et al., 2004
4D in vivo video microscopy showed that in wild-type cells, a subset of peroxisomes partitioned to the emerging bud, whereas the peroxisomes that remained in the mother cell retained fixed cortical positions. The newly inherited peroxisomes tend to concentrate at the sites of active growth inside the bud. Before cytokinesis, subsets of peroxisomes from both the mother cell and the bud redistribute to the neck region, whereas the remaining peroxisomes remain anchored to the cortices of the mother cell and bud.
Peroxisomes of inp1Δ cells displayed increased mobility relative to peroxisomes of wild-type cells and were never observed to be static. Moreover, in inp1Δ cells, there was no delay as compared with wild-type cells in the passage of peroxisomes to the emerging bud, except in those cells containing greatly enlarged peroxisomes. Therefore, Inp1p is not directly involved in the movement of peroxisomes between mother cell and bud, presumably along actin tracks. How then might Inp1p function in peroxisome inheritance? An interesting feature of the dynamics of peroxisomes in cells lacking Inp1p is that the entire peroxisome population in the mother cell first clusters at the presumptive bud site and then enters the bud, thereby depleting the mother cell of peroxisomes. At times, peroxisomes were observed that failed to be delivered to the growing bud, but they also appeared to be unattached to the mother cell cortex, performing chaotic movements within the mother cell. On occasion, peroxisomes, after having passed to the bud, returned deep into the interior of the mother cell, a phenomenon never observed in wild-type cells. Actin as a whole is apparently normal in inp1Δ cells (unpublished data), and thus a major reorganization of the actin cytoskeleton cannot explain why inp1Δ cells exhibit defects in peroxisome inheritance. In inp1Δ cells, peroxisomes fail to be actively retained in either the mother cell or the bud, which results in the disruption of the ordered vectorial process of peroxisome segregation during cell division. The movements of peroxisomes from buds to mother cells could be explained by proposing that peroxisomes delivered to the bud in inp1Δ cells have a decreased affinity for a structure that retains peroxisomes within the bud, with the possibility that some peroxisomes actually elude the anchoring mechanism completely. Because the return of newly inherited peroxisomes usually occurred after their performance of the characteristic movements of peroxisomes in the bud observed in wild-type cells, including the initial clustering of peroxisomes at the bud tip, we would predict that other factors must also play a role in maintaining newly inherited peroxisomes in the bud, at least in the early stages. The overproduction of Inp1p results in the retention of peroxisomes in the mother cell at fixed cortical positions and prevents the distribution of a subset of peroxisomes to the growing bud. Occasionally, one peroxisome would be delivered to the bud and, after performing the usual movements in the bud, would gain a fixed position at the bud cortex. The fact that when overproduced Inp1p assumes a cortical distribution in glucose-grown cells containing few peroxisomes strengthened our conclusion that Inp1p acts to tether peroxisomes to anchoring structures localized to the periphery of cells. All in all, our data reveal a major role for Inp1p in tethering peroxisomes to anchoring structures in both mother cell and bud during cell division.
Evidence for Inp1p being regulated during the cell cycle suggests that peroxisome inheritance is tightly controlled by the cell. Increased amounts of Inp1p at certain stages of the cell cycle might be required to ensure the retention of peroxisomes in both mother cell and bud. Inp1p might increase in amount only on a subset of peroxisomes that become prone to anchoring at the cell cortex. Alternatively, Inp1p might be fairly equally distributed on all peroxisomes, and other regional regulatory mechanisms and molecules could themselves act through Inp1p to modulate the anchoring of peroxisomes to the cell cortex. The oscillation of Inp1p levels during the cell cycle correlates with the oscillation of INP1
mRNA levels during the cell cycle (Spellman et al., 1998
), suggesting that the INP1
gene is subject to cell cycle regulatory control. It is noteworthy that Inp1p is predicted to contain a PEST sequence (a potential signal for rapid protein degradation) between amino acids 279 and 362 (Rechsteiner and Rogers, 1996
). Whether this PEST sequence functions in the degradation of Inp1p during the cell cycle awaits future experimentation.
A model for Inp1p function in partitioning peroxisomes between mother cell and bud is presented in . A subset of peroxisomes is transported to the bud by a process dependent on Myo2p (Hoepfner et al., 2001
), whereas the remaining peroxisomes are retained within the mother cell on a cortical anchor. The peroxisomal peripheral membrane protein Inp1p would link the peroxisome to the cortical anchor. It is noteworthy that overproduction of Inp1p led to a distinctly enhanced cortical distribution of peroxisomes in cells. Whether a given peroxisome will be delivered to the bud or retained in the mother may depend on a tug-of-war between Inp1p and Myo2p. Accordingly, both under- and overproduction of Inp1p would lead to impairment of normal peroxisome inheritance. Once peroxisomes are delivered to the bud, they are prevented from returning to the mother cell. Inp1p also appears to play a role also in retaining peroxisomes within the bud, probably by attaching peroxisomes to cortical anchoring structures present in the bud. Actin structures do not appear to play a role in the Inp1p-dependent anchorage of peroxisomes to the cell cortex, because the treatment of cells overproducing Inp1p with Lat A did not lead to the detachment of immobilized peroxisomes. Moreover, we did not observe a colocalization between the Sac6p-containing actin patches and peroxisomes (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200503083/DC1
Figure 10. A model for Inp1p function in peroxisome retention. Peroxisomes move along polarized actin cables in a Myo2p-dependent manner from mother cell to bud. Concomitantly, a subset of peroxisomes is retained within the mother cell. Inp1p acts to link peroxisomes (more ...)
The phenotype of reduced numbers of enlarged peroxisomes seen for inp1
Δ cells could conceptually arise only as a consequence of unbalanced partitioning of peroxisomes during cell division. However, the interactions of Inp1p with Pex25p, Pex30p, and Vps1p, which have all been shown previously to influence peroxisome division, support a role for Inp1p in peroxisome division. Thus, Inp1p seems to have a dual role in the division and the inheritance of peroxisomes in S. cerevisiae
. How might these two functions be related? Other proteins are known to influence both the morphology of organelles and their distribution. Mdm10p (Sogo and Yaffe, 1994
), Mdm12p (Berger et al., 1997
), and Mmm1p (Burgess et al., 1994
) are mitochondrial outer membrane proteins that affect mitochondrial shape and segregation. Mutation of any one of these proteins results in the presence of giant, spherical mitochondria that exhibit defects in partitioning at cell division. Recent studies (Boldogh et al., 2003
) have indicated that these proteins form a complex that connects the minimum heritable unit of mitochondria (mtDNA and mitochondrial membranes) to actin, therefore functioning as a mitochondrial counterpart to the kinetochore or the “mitochore.” These proteins affect the retention of mitochondria within the mother cell (Yang et al., 1999
) and also Myo2p-independent mitochondrial movement (Boldogh et al., 2001
In closing, we have presented evidence demonstrating that the peroxisomal peripheral membrane protein, Inp1p, is directly implicated in the inheritance of peroxisomes in S. cerevisiae. Inp1p acts as a peroxisome-retention factor, tethering peroxisomes to putative anchoring structures within the mother cell and bud. Inp1p is the first peroxisomal protein shown to be involved in the inheritance of peroxisomes.