Functional involvement of Rab1A in microtubule-dependent anterograde melanosome transport in melanocytes

Abstract

SummaryMelanosomes are transported to the cell periphery of melanocytes by coordination between bidirectional microtubule-dependent movements and unidirectional actin-dependent movement. Although both the mechanism of the actin-dependent melanosome transport and the mechanism of the microtubule-dependent retrograde melanosome transport in mammalian skin melanocytes have already been determined, almost nothing is known about the mechanism of the microtubule-dependent anterograde melanosome transport. Small GTPase Rab proteins are common regulators of membrane traffic in all eukaryotes, and in this study we performed genome-wide screening for Rab proteins that are involved in anterograde melanosome transport by expressing 60 different constitutive active (and negative) mutants, and succeeded in identifying Rab1A, originally described as a Golgi-resident Rab, as a prime candidate. Endogenous Rab1A protein was found to be localized to mature melanosomes in melanocytes, and its functional ablation either by siRNA-mediated knockdown or by overexpression of a cytosolic form of Rab1A-GTPase-activating protein/TBC1D20 induced perinuclear melanosome aggregation. The results of time-lapse imaging further revealed that long-range anterograde melanosome movements were specifically suppressed in Rab1A-deficient melanocytes, whereas retrograde melanosome transport occurred normally. Taken together, these findings indicate that Rab1A is the first crucial component of the anterograde melanosome transport machinery to be identified in mammalian skin melanocytes.

Small GTPase Rab2B and Its Specific Binding Protein Golgi-associated Rab2B Interactor-like 4 (GARI-L4) Regulate Golgi Morphology

“…Actually, four members of the Rab3 subfamily (Rab3A/B/C/D) are known to have redundant roles in mouse survival (39), and two members of the Rab8 subfamily (Rab8A/B) have compensatory roles in the apical transport of epithelial cells (40). Recent accumulating evidence, however, indicates that the A and B isoforms of Rabs have different functions in certain membrane trafficking events and different subcellular localizations even within the same cell type (41)(42)(43)(44)(45)(46)(47). One good example is the Rab27 subfamily in which Rab27A and Rab27B differently regulate the exocytosis of certain types of lysosome-related organelles, e.g.…”

ARHGEF10 directs the localization of Rab8 to Rab6-positive executive vesicles

“…Fragments encoding mouse ARHGEF10 L548A were amplified by PCR and subcloned into pCMVMyc vector. pEGFP-C1 Rab5a, Rab6a, Rab7, Rab8a, Rab11a and Rab27a were kindly provided by Mitsunori Fukuda (Tohoku University, Aobayama, Aoba-ku, Sendai, Miyagi, Japan) (Itoh et al, 2006;Tsuboi and Fukuda, 2006;Tamura et al, 2009;Ishida et al, 2012;Kobayashi and Fukuda, 2012). pNPY-Venus-N1 was gift from Atsushi Miyawaki (RIKEN, Hirosawa, Wako City, Saitama, Japan) (Nagai et al, 2002).…”

Rab5 Is a Novel Regulator of Mast Cell Secretory Granules: Impact on Size, Cargo, and Exocytosis

“…Constitutively negative (CN) and constitutively active (CA) Rab mutants were prepared as described previously (26,27) and subcloned into the pEGFP-C1 vectors (28). The nomenclature of the Rabs is according to Itoh et al (26).…”