Abstract
The mechanism(s) by which Rab GTPases are specifically recruited to distinct intracellular membranes remains elusive. Here we used Rab27a localisation onto melanosomes as a model to investigate Rab targeting. We identified the α1 subunit of Na+,K+-ATPase (ATP1a1) as a novel Rab27a interacting protein in melanocytes and showed that this interaction is direct with the intracellular M4M5 loop of ATP1a1 and independent of nucleotide bound status of the Rab. Knockdown studies in melanocytes revealed that ATP1a1 plays an essential role in Rab27a-dependent melanosome transport. Specifically, expression of ATP1a1, like the Rab27a GDP/GTP exchange factor (Rab3GEP), is essential for targeting and activation of Rab27a to melanosomes. Finally, we showed that the ability of Rab27a mutants to target to melanosomes correlates with the efficiency of their interaction with ATP1a1. Altogether these studies point to a new role for ATP1a1 as a regulator of Rab27a targeting and activation.
Highlights
Rab GTPases are essential regulators of intracellular membrane trafficking [1]
These observations indicate that it is highly likely that HTF-Rab27a proteins expressed in Rab27a null melanocytes interact with a group of proteins similar to that encountered by the endogenous Rab27a in wild-type melanocytes
HTF-Rab27a proteins were purified from these cell lines by sequential immunoprecipitations and co-purified proteins were visualised by silver staining (Figure 1 C) before identification using nano-liquid chromatography mass spectrometry
Summary
Rab GTPases are essential regulators of intracellular membrane trafficking [1]. Key to this activity is their ability to associate with specific membrane compartments within the cell [2]. A number of mechanisms have been postulated including the involvement of the C-terminal hypervariable domain [4,5,6,7], Rab effector binding [8], GDI displacement factors [9,10,11] and RabGEFs [12,13,14,15], all of which has been summarised in a recent review [16]. Rab27a associates with melanosomes and recruits its effector Melanophilin (Mlph/Slac2-a/Exophilin 5), which binds the actinbased motor protein MyosinVa (MyoVa). For further details on melanogenesis the author directs the reader to the following reviews [25,26,27]
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