Melanosomes are melanin-containing organelles in melanocytes, and they are responsible for skin and hair pigmentation in mammals. The intracellular distribution of melanosomes is mainly determined by the balance between their anterograde transport on actin filaments and retrograde transport on microtubules. Although we have shown previously that melanoregulin (Mreg) and Rab36 serve as cargo receptors on melanosomes for retrograde transport, their knockdown does not completely inhibit retrograde melanosome transport, suggesting the existence of an additional cargo receptor(s) in melanocytes. In this study, we investigated the possible involvement of an atypical large Rab, Rab44, which also contains EF-hand domains and a coiled-coil domain, in retrograde melanosome transport in mouse melanocytes (Rab27A-deficient melan-ash cells). Our results showed that Rab44 localizes on mature melanosomes through lipidation of its C-terminal Rab-like GTPase domain, and that its knockdown results in suppression of retrograde melanosome transport. Additionally, our biochemical analysis indicated that Rab44 interacts with the dynein–dynactin motor complex via its coiled-coil-domain–containing middle region. Since simultaneous depletion of Rab44, Mreg, and Rab36 resulted in almost complete inhibition of retrograde melanosome transport, we propose that Rab44 is the third cargo receptor. We also showed that the N-terminal region of Rab44, which contains EF-hand domains, is required for both retrograde melanosome transport and its Ca2+-modulated activities. Our findings indicated that Rab44 is a third melanosomal cargo receptor, and that, unlike other cargo receptors previously described, its transport function is regulated by Ca2+.

dynein

EF-hand

melanocyte

large Rab

melanosome

membrane trafficking

organelle

Rab44

retrograde transport

small GTPase

α-melanocyte-stimulating hormone

constitutively negative

glyceraldehyde-3-phosphate dehydrogenase

glutathione S-transferase

horseradish peroxidase

lysosome-related organelles

pigment dispersion distance

Rab-interacting lysosomal protein

© 2022 THE AUTHORS. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology.