In this study we used a cell culture melanosome transport assay to define critical functional domains of the Mlph protein. In contrast to previous studies in which overexpression of Mlph mutants was observed in wild-type melanocytes, our approach of using Mlph-null cells provides clearer information regarding the formation of the tripartite Rab27a:Mlph:MyoVa complex and in particular the contribution of Mlph domains to melanosome transport because the interpretation of results is not complicated by the presence of wild-type Mlph (Kuroda and Fukuda 2003). This system also allows the contribution of individual Mlph interacting proteins to be examined by using mutant Mlph proteins that specifically disrupt each protein:protein interaction and then assaying their function in a cellular environment.
Using the cell culture melanosome transport assay, we reached several unexpected conclusions regarding Mlph domains and interacting partners. Our data indicate that MyoVa recruitment to melanosomes and recovery of leaden
melanosome transport defects cannot be uncoupled. Moreover, we find that a coiled-coil forming region of the Mlph C-terminus tail (aa 440-483) is essential for this function ( and ). Our results using the melanosome transport assay and an in vitro binding assay indicate that the coiled-coil contributes significantly to the ability of Mlph to recruit MyoVa to the melanosome membrane. This is unexpected because other in vitro studies showed that this Mlph coiled-coil region does not contribute to interaction with or activation of actin-dependent ATPase activity of MyoVa and indicated that R27BD together with MBD should allow recruitment of MyoVa to melanosomes (Nagashima et al., 2002
; Li et al., 2005
). Interestingly, it appears that the coiled-coil region alone has little affinity for MyoVa and only participates in binding when present in combination with the MBD (). Furthermore, this coiled-coil sequence is similar to the corresponding region of the Rab27 and MyoVa/VIIa-linking protein MyRIP, and our data indicate that this region also performs the same function in activation of MyoVa ( and ). Although there is little primary sequence conservation between the cargo-binding tails of these different myosin motors, the conservation of the coiled-coil structure in MyRIP suggests that it may contribute to interaction with and/or activation of MyoVIIa and provide indications that a similar mechanism allows the activation of different myosin motors by cargo-specific proteins.
Leading from this, our second conclusion is that the integrity of the C-terminus ABD of Mlph and its ability to associate with actin, EB1 (and MyoVa, this study) appear to be dispensable for melanosome transport in melanocytes. Previously, Mlph aa 490-590 was suggested to form a binding site for EB1, whereas basic amino acids K493, R495, R496, and K497 allow interaction with filamentous actin (Fukuda 2002
; Wu et al., 2005
). It is suggested that Mlph interaction with actin might enhance the processivity of the MyoVa motor activity, whereas EB1 is proposed to allow transport of Mlph to the periphery of melanocyte cytoplasm before Rab27a-Mlph-MyoVa complex formation, via transport at the + tips of growing microtubules. Our data argue that these interactions are likely to fulfill an ancillary function in melanosome transport as rescue of leaden
melanosome transport defects occurs with efficiency similar to that of wild-type when Mlph1-483 or MlphKA are expressed ( and ). Alternatively, these interactions may play an important role in aspects of melanosome transport and pigmentation in vivo that may not be measured using this cell culture assay.
A third unexpected conclusion of our study is that the region of the MBD proposed to be important for interaction with the MyoVa globular tail (GTBD aa 150-266) makes a nonessential contribution to Mlph function. Evidence for this is that deletion of this region of Mlph does not affect the ability of the MlphΔGTBD mutant to rescue melanosome transport defects ( and ). However, these nonessential pieces of Mlph, GTBD and Mlph aa 490-590, appear to fulfill a role in stabilization of the core MyoVa-binding region (300-484 aa) because their presence enhances the level of MyoVa recruited to melanosome, and the presence of either of these pieces is essential for functional rescue of melanosome transport (, , , and ).
In addition to these unexpected conclusions, our assay allowed us to confirm the role of other parts of Mlph predicted by earlier studies and to confirm the significance of the contribution that these parts play in Mlph function. First, we confirm that the R27BD allows association with Rab27a-GTP on melanosomes (). Specifically we show that SHD1 and SHD2 form a minimal R27BD, whereas the ZnF is dispensable for this interaction. This is consistent with the fact that all Rab27 binding Slp and Slac2 family proteins contain SHD1 and SHD2 but not ZnF and suggests that the in situ requirements for interaction of Rab27a and Mlph are more stringent than those reported using recombinant proteins (Fukuda et al., 2002
). Second, our results underline the importance of the EFBD of Mlph because mutants MlphΔEFBD and MlphEA are unable to restore wild-type melanosome transport to leaden
melanocytes ( and ). Thirdly, our biochemical data suggest that truncated Mlph proteins do not interact with MyoVa with the same efficiency as wild-type; however, those containing the two essential domains, EFBD and coiled-coil, together with either of the other regions, GTBD and extreme C-terminus, allow sufficient levels of MyoVa binding to allow functional rescue. This raises the possibility that there is some level of functional redundancy between the latter domains. In addition, it is possible that association of MlphMBD with MyoVa may reveal MyoVa binding site(s) within the coiled-coil and extreme C-terminus of Mlph as these regions alone (Mlph400-500 and Mlph400-590) are unable undertake interaction with MyoVa when isolated from MBD (A; our unpublished observations).
Finally, our study addressed the relationship between recruitment of melanosomal MyoVa and recovery of melan-ln melanosome transport defects. Our finding shows conclusively that Mlph constructs that are able to recruit MyoVa (~20–40% of wild-type levels) to melanosomes also allowed melanosomes to be retained in peripheral dendrites. Melanosome transport and in vitro binding assays confirm that both EFBD and coiled-coil regions together with either GTBD or the extreme C-terminus allow this to be achieved (, , and ). This striking correlation between MyoVa recruitment and melanosome transport rescue suggests that the critical event in peripheral melanosome transport is the formation of the tripartite complex. One possibility to explain our findings is that melanosomal recruitment is somehow coupled with a conformational change in MyoVa, as has been recently proposed for its functional activation in high Ca2+
(Krementsov et al., 2004
; Li et al., 2004
; Wang et al., 2004
). Unfortunately, the activation status of MyoVa cannot be probed directly using this assay, and future studies should address this possibility using assays that measure the motor activity of MyoVa in vitro.
In conclusion, our data clearly implicate the coiled-coil region of Mlph in MyoVa binding and activation. The structural and functional conservation of this motif in MyRIP opens the possibility that a conserved mechanism for activation of MyoVa and MyoVIIa may exist. Future studies should be directed toward understanding the structural mechanism by which this is achieved.