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 a1 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.