Characterization of organelle-specific cargo adaptors, which directly link cargoes to myosin V motors, revealed that organelle transport occurs via direct regulation of cargo adaptor proteins (Moore and Miller, 2007
; Peng and Weisman, 2008
; Fagarasanu et al., 2009
). For example, initiation of vacuole movement requires Cdk1-dependent phosphorylation of the adaptor Vac17 (Peng and Weisman, 2008
), which promotes Vac17 binding to Myo2 and vacuole movement. Vac17 regulation is also required for termination of vacuole movement (Tang et al., 2003
). When the vacuole reaches the bud, a pathway that utilizes the Vac17 PEST sequence promotes the degradation of Vac17. In addition, Vac17 expression is coordinated with the cell cycle and peaks when the vacuole moves into the bud (Tang et al., 2003
; Peng and Weisman, 2008
Multiple modes of regulation of Vac17 suggested that cargo adaptors regulate cargo movement, whereas the motor provides an inert platform for cargo binding. In support of this postulate, two cargo adaptors, Vac17 and the secretory vesicle-specific Rabs Ypt31/32, bound distinct sites on Myo2. Thus, it was assumed that binding of each Myo2 adaptor would be independently regulated.
Evidence that the CBD of myosin V plays a role cargo selection came from discoveries that Xenopus laevis
myosin V and yeast Myo2 undergo reversible phosphorylation (Rogers et al., 1999
; Karcher et al., 2001
; Legesse-Miller et al., 2006
). Moreover, phosphorylation of the myosin V CBD in Xenopus
oocytes regulates cargo interactions. Thus, phosphorylation of the myosin V CDB may facilitate the attachment or detachment of cargoes.
Further evidence that the CBD of Myo2 may regulate cargo selection was the finding that the Inp2 and Rab GTPase binding sites overlap (Fagarasanu et al., 2009
). Kar9 and Ypt11 also interact with residues at this region (). Similarly, Mmr1 binds to a second site, which overlaps with the Vac17 binding site (). Overlap of eight cargo adaptors at two sites suggested that the overlap has a function.
While this paper was in revision, a new study showed that Pex19 binds to Myo2 at residue I1232, adjacent to the Mmr1/Vac17 binding site, and at I1440, adjacent to the Rab GTPase/Inp2/Kar9 site (Otzen et al., 2012
). Thus, the complexity of interactions between cargo adaptors is even greater than described here.
We postulated that physical constraints allow only Mmr1 or Vac17 to occupy Myo2 at a given time. Indeed, this overlap contributes to the regulation of the inheritance of mitochondria and vacuoles (). One possibility was that competition between Mmr1 and Vac17 provided a temporal order for the inheritance of these organelles. However, mitochondria and vacuoles move into the bud at similar times (). Moreover, either the vacuole or mitochondria moved first, or both crossed the mother–bud neck simultaneously. Thus, Mmr1 and Vac17 have equal access to Myo2.
Despite similar access to Myo2, Mmr1 and Vac17 compete with each other in vivo and in vitro. The major significance of this competition is regulation of the volume of inherited vacuoles and mitochondria. Little is known about the regulation of organelle volume (Chan and Marshall, 2010
). It had been assumed that organelle volume is regulated via a combination of organelle biogenesis and autophagy/turnover. Our experiments reveal an additional determinant based on the competition between cargo adaptor proteins for Myo2. A mutant that blocks mitochondria inheritance has an increased vacuole volume in the bud. Conversely, a mutant that blocks vacuole inheritance has an increased mitochondrial volume in the bud. This greater volume of mitochondria or vacuoles persists and is observed in large budded and unbudded cells. Thus, competition between Mmr1 and Vac17 for access to Myo2 plays a major role in regulating the volume of mitochondria and vacuoles ().
Figure 8. Model for competition between Vac17 and Mmr1 for access to Myo2. (A) Myo2 transports vacuoles (V) and mitochondria (M). Mmr1/Myo2 localizes to the leading portion of the mitochondrial tubule in the mother near the mother–bud neck and on mitochondria (more ...)
Both mitochondria and vacuoles undergo retrograde movement across the mother–bud neck. One possibility is that Myo2 stochastically releases cargoes through a loss of Myo2 attachment to the cargo adaptor. If true, there would be less rocking in mutants that affect only one cargo adaptor. Indeed, in both the myo2-D1297N and myo2-I1308 mutants, there are fewer retrograde movements of the inherited organelle. One interpretation is that the probability of reinitiation of Myo2 binding to Vac17 (for myo2-I1308A) or Mmr1 (for myo2-D1297N) is higher when there is not competition from the other cargo adaptor.
Mutants with defects in moving mitochondria or vacuoles often had portions of each organelle poised in the mother at the mother–bud neck. This suggests that attachment of the organelle to Myo2 is not completely blocked. Furthermore, a higher level of force might be needed to move organelles through the mother–bud neck. Perhaps fewer motors are required to bring a portion of the organelle to the mother–bud neck, and additional motors are required for movement through the mother–bud neck. The environment in the neck may contribute to the rocking motion.
Eventually, stable pools of mitochondria and vacuoles are established in the bud. Tethering proteins likely anchor these organelles. Mmr1 and Ypt11, as well as ERMES tethering of mitochondria to the endoplasmic reticulum, may perform this function for mitochondria. Similarly, there are likely tethers for the vacuole. Interestingly, we observed that overexpression of either VAC17 or MMR1 overcomes a tethering mechanism in the mother cell for vacuoles or mitochondria, respectively ().
That mitochondria and vacuoles move across the mother–bud neck at similar times and that Mmr1 and Vac17 appear to have equal access to a common binding region on Myo2 suggest that the cell cycle–dependent regulation of Vac17 and Mmr1 may be similar. Mmr1 may be the target of the same Cdk1–cyclin complexes that target Vac17 (Peng and Weisman, 2008
); Mmr1 has seven Cdk1 consensus sites. Along similar lines, we predict that detachment of Mmr1 from Myo2 may use a similar mechanism to that observed with Vac17. Vac17 detachment from Myo2 requires the Vac17 PEST sequence. Similarly, Mmr1 contains two predicted PEST sequences (Fig. S2). Moreover, like Vac17, Myo2 mutations that block mitochondria inheritance result in an elevation of Mmr1.
Determination of the functional significance of the overlap of six cargo adaptors at the Rab GTPase binding site will be challenging. In addition to the Rab GTPase Ypt11 and the secretory vesicle Rab GTPases Ypt31/32 and Sec4, Kar9 and Inp2 bind at this region. Moreover, all cargo adaptor binding sites may modulate each other through structural changes in the Myo2 CBD.
Binding of the Rab GTPases Ypt31/32 and Sec4 may impact the Sec15 binding site and vice versa (). Loop H in the CBD structure contains a Sec15-binding residue and connects to helix 9, which has several residues critical for interaction with Kar9, Inp2, and the Rab GTPases. Helix 12 contributes residues to Sec15 binding, and loop L, which extends from this helix, contains residues important for Kar9 and Inp2 binding. Thus, binding of Inp2, Kar9, and/or Rab GTPases may change the Sec15 binding site or vice versa. This may be critical to the regulation of Myo2 attachment to secretory vesicles.
Similarly, the Mmr1/Vac17 binding region on helix 6 is connected to loop F, which contains residues in the Rab GTPase/Kar9/Inp2 binding site. Thus, binding of a Rab or other proteins to this region may affect the binding of Vac17 and Mmr1 and vice versa. In support of this hypothesis, mutation of myo2-L1301 on helix 6, which disrupts Myo2 interaction with Mmr1 and Vac17, also disrupts the binding of Kar9 and Smy1, which suggests that this mutation causes a distant conformational change of the surface residues in the Kar9-binding region.
Secretory vesicles are the only known essential cargo of Myo2. Thus, it is tempting to speculate that Ypt31/32 and Sec4, required for secretory vesicle interaction with Myo2, may have priority to occupy the CBD. Thus, cargo transport may be coordinated in part through structural changes in the myosin V CBD that occur through long distance communication between each of the adaptor binding sites.