Most intracellular movement of organelles in S. cerevisiae
is powered by the class V myosin motor Myo2p. Interestingly, each Myo2p cargo displays Myo2p-dependent motility at a distinct time in the cell cycle. Also, the ultimate destination of Myo2p-driven transport is specific for each type of organelle (Fagarasanu et al., 2006b
; Weisman, 2006
). Therefore, Myo2p's attachment to and detachment from organelles are independently regulated for each type of organelle.
It is crucial to understand the structural basis for Myo2p's association with its cargoes to elucidate the regulatory mechanisms that allow it to move multiple cargoes to distinct places at different times. The regions on the surface of Myo2p required for binding two of its cargoes, namely the vacuole and secretory vesicles, have been identified (Schott et al., 1999
; Catlett et al., 2000
). These regions were found to be distant from one another and simultaneously exposed on the surface of the globular tail of Myo2p (Pashkova et al., 2006
), suggesting that the tail does not have a major role in regulating cargo binding. Rather, the availability of cargo-specific receptors must dictate the timing of organelle attachment to Myo2p (Fagarasanu et al., 2006b
; Pashkova et al., 2006
; Weisman, 2006
). In this study, we defined the surface region of the Myo2p tail devoted to binding peroxisomes and showed that this region is distinct from the region previously identified to bind vacuoles but partially overlaps the region that binds secretory vesicles. Recently, two surface residues that participate in vacuole binding were found to also function in the Myo2p-driven transport of mitochondria (Altmann et al., 2008
). Therefore, it is likely that the yet unidentified mitochondrion-binding region of Myo2p overlaps its vacuole-binding region. These findings challenge the currently held view that the spatial segregation of various organelle-binding regions is an important feature of Myo2p, allowing it to function as a scaffold that exposes all its cargo-binding sites at the same time while still avoiding competition for the transport of different cargoes (Weisman, 2006
). Overlap in the regions on the Myo2p surface specialized in binding different organelles suggests that different types of organelle could potentially compete with one another for access to Myo2p. This capacity for steric exclusion in myosin–organelle interactions would impose a tight temporal regulation on the activities of organelle-specific Myo2p receptors during the cell cycle and result in different receptors acting at different times in the inheritance of their specific organelles.
In this study, we elucidated the spatial and temporal parameters that contribute to the regulation of Inp2p, the peroxisome-specific receptor for Myo2p. The levels of Inp2p fluctuate during the cell cycle, being maximal when peroxisome inheritance occurs and decreased later in the cell cycle when about half of the peroxisomes have been delivered to the bud (Fagarasanu et al., 2006a
). To gain insight into how Inp2p is regulated during the cell cycle, we followed the dynamics of Inp2p when peroxisome transfer to the bud was prevented or delayed using mutants of Myo2p that are defective in binding and transporting peroxisomes. Cells harboring such Myo2p mutants as the sole copy of Myo2p produced buds devoid of peroxisomes but still progressed normally through the cell cycle, thereby effectively dissociating the two processes of peroxisome segregation and cell cycle progression. We found that under these conditions, Inp2p levels were increased, indicating that the cellular abundance of Inp2p is not intrinsically linked to the cell cycle but rather determined by the distribution of peroxisomes in the dividing cell, i.e., by organelle positioning–specific cues. Moreover, in contrast to wild-type cells in which the Inp2p signal is highly polarized toward the bud, most peroxisomes in the mother cells of myo2
mutants contained increased amounts of Inp2p. Therefore, the misplacement of peroxisomes caused by point mutations in the Myo2p tail influenced both the localization and levels of Inp2p. These data are consistent with a scenario in which the synthesis of Inp2p in the mother cell and its initial accumulation on a subset of peroxisomes precede its Myo2p-driven transport along with peroxisomes to the bud. In the bud, Inp2p–Myo2p transport complexes are eventually disassembled through the regulated degradation of Inp2p, releasing the transferred peroxisomes from Myo2p. Accordingly, when the association of peroxisomes with the translocation machinery is disrupted, as is the case for the myo2
mutants with compromised peroxisome inheritance, Inp2p is protected from the bud-specific proteolytic turnover. A degradation of Inp2p in the bud to terminate peroxisome motility agrees with current models for the inheritance of other organelles, such as yeast vacuoles. Vac17p, the vacuole-specific receptor for Myo2p, was proposed to be degraded in the bud after the transfer of vacuolar membranes into the bud (Tang et al., 2003
Our data are also consistent with the existence of additional regulatory mechanisms that survey the intracellular distribution of Inp2p (). If the increase in Inp2p levels in the myo2
peroxisome inheritance mutants resulted exclusively from a lack of Inp2p degradation at its destination, i.e., in the bud, one would expect approximately the same number of peroxisomes to contain detectable levels of Inp2p in the wild-type and myo2
mutant strains. However, this is not the case, as the inheritance mutant myo2-Y1483A
displays increased amounts of Inp2p on most, if not all, peroxisomes in the mother cell. These findings strongly suggest the existence of a regulatory feedback mechanism from bud to mother cell that causes Inp2p to accumulate aberrantly on peroxisomes in the mother cell upon disruption of peroxisome inheritance (). Because the levels of INP2
mRNA are essentially the same in wild-type cells and cells of the myo2-Y1483A
mutant, this proposed feedback mechanism must operate posttranscriptionally, most likely through degradation machinery acting on Inp2p. Cellular surveillance mechanisms that monitor peroxisome partitioning could be envisioned to trigger the degradation of Inp2p in response to efficient peroxisome inheritance (). If the Inp2p degradation machinery is present in both mother cell and bud, its activation would not only cause the release of transferred peroxisomes from Myo2p but would also prevent new recruitments of additional peroxisomes from the mother cell. Thus, proteolytic degradation of the receptor/adaptor protein in the myosin-capturing complex could represent an effective mechanism not only for depositing a moving organelle at its proper destination (Weisman, 2006
; Fagarasanu and Rachubinski, 2007
; Li and Nebenführ, 2008
) but also for terminating organelle inheritance.
Figure 7. A model for Inp2p regulation. At the beginning of the cell cycle, Inp2p is loaded onto all peroxisomes. Those peroxisomes with more Inp2p have a greater probability of being carried by Myo2p into the bud. The presence of peroxisomes in the bud prevents (more ...)
The presence of an Inp2p signal on most peroxisomes in the mother cell was also observed when peroxisome transfer to the bud was prevented by another means, i.e., by overproduction of Inp1p. This result clearly showed that cells alter Inp2p distribution and levels in a compensatory manner in an attempt to reestablish correct peroxisome placement irrespective of the cause of the defect in peroxisome segregation. This result also demonstrated that it is not the lack of engagement of Inp2p by the Myo2p motor that leads to the changes in Inp2p distribution observed in the myo2-Y1483A mutant cells.
Support for this model comes from several different observations. First, the highly polarized Inp2p-GFP signal along the cell division axis in wild-type cells not only demonstrates the selectivity of Myo2p in carrying those peroxisomes that have increased amounts of Inp2p to the growing bud (Fagarasanu et al., 2006a
) but also shows that Inp2p is not degraded as soon as it is exposed to the bud environment. The degradation of Inp2p is probably influenced by the extent of peroxisome transfer and is thus triggered after a sufficient number of peroxisomes have been partitioned to the daughter cell. Second, although the Inp2p-GFP signal in wild-type cells is not uniformly distributed among different peroxisomes, most likely all peroxisomes can acquire Inp2p, but its levels on some peroxisomes may be below the threshold of microscopic detection. Evidence to support this comes from the observation that in cells lacking the peroxisomal anchor protein Inp1p, wherein all peroxisomes have lost their ability to remain attached to the cell periphery, the entire peroxisome population is eventually transferred to daughter cells (Fagarasanu et al., 2005
), presumably in an Inp2p- and Myo2p-dependent manner (Fagarasanu and Rachubinski, 2007
). This shows that in the myo2
peroxisome inheritance mutants, the distribution of Inp2p on peroxisomes is probably similar to that found in wild-type cells, but the amount of Inp2p is increased. The delay in peroxisome partitioning in these mutants is sufficient for Inp2p levels on all peroxisomes to increase above the detection threshold, allowing for direct monitoring of how peroxisome inheritance feeds back onto Inp2p levels in the mother cell. Third, we observe a decrease in the Inp2p-GFP signal on peroxisomes in the mother cell in an inheritance mutant when peroxisomes are segregated to the bud. This suggests an inverse relationship between Inp2p levels in the mother cell and the efficiency of peroxisome transfer to the bud. Fourth, the levels of Inp2p in a peroxisome inheritance mutant, while being significantly increased, still oscillate in the cell cycle with the same pattern observed for wild-type cells. Therefore, cell cycle–related fluctuations in Inp2p levels occur irrespective of the efficiency of peroxisome inheritance. This provides further proof that Inp2p turnover can occur both in the mother cell and the bud (). The degradation complex might be present in the cytosol or even on the peroxisomal membrane, but it appears that its activity is determined by both peroxisome positioning and cell cycle cues.
The presence of only one greatly enlarged peroxisome in cells lacking Vps1p facilitates the analysis of Inp2p dynamics because it leads to the accumulation of all Inp2p molecules in the membrane of a single peroxisome. Under these circumstances, it was evident that Inp2p-GFP initially sampled the entire peroxisome, with its leading edge containing higher levels of Inp2p. However, later in the cell cycle, only that part of the enlarged peroxisome present in the bud preserved Inp2p, and no Inp2p-GFP could be detected in the mother cell. In contrast, in cells lacking Vps1p and expressing the myo2-Y1483A mutation, the peroxisomes in mother cells contained detectable amounts of Inp2p. This observation is consistent with our model in which the peroxisomes that initially accumulate Inp2p, or in the case of vps1Δ cells, the region of the tubule that initially accumulates Inp2p, are delivered to the bud first and ultimately trigger the down-regulation of Inp2p in the peroxisomes remaining in the mother cell ().
Feedback regulation of Inp2p could be provided by a posttranslational modification, such as phosphorylation, that would make Inp2p susceptible to degradation. In this study, we show that Inp2p is a phosphoprotein whose level of phosphorylation correlates with the cell cycle, being more pronounced at the start and end of the cell cycle. Notably, Vac17p was shown to be activated in the mother cell by phosphorylation by Cdk1p (Peng and Weisman, 2008
) and in the bud by Cla4p and Ste20p, an event that leads to its turnover (Bartholomew and Hardy, 2009
). The existence of two temporally distinct phosphorylation events of Inp2p hints at the possibility of successive steps of checkpoint-dependent activation and inactivation of Inp2p, such as its preparation for binding to Myo2p early in the cell cycle and its preparation for degradation late in the cell cycle. However, because the times at which the phosphorylated forms of Inp2p appear and disappear are the same in wild-type cells and cells of the peroxisome inheritance mutant myo2-Y1483A
, the timing of phosphorylation of Inp2p is probably coupled to the cell cycle and not related to intracellular peroxisome placement.
In closing, we have shown that Inp2p, the peroxisome-specific receptor for Myo2p, is subject to both spatial and temporal regulation. Our findings point to the existence of cell cycle–dependent and organelle positioning–dependent mechanisms that control the activity and levels of organelle-specific receptors for the molecular motors that drive the intracellular motility of different membrane-bound compartments. Given that the different types of organelles compete for access to these motors, cells have developed multiple levels of regulation for their organelle-specific receptors so as to ultimately achieve an equal distribution of compartments between mother and daughter cells.