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Vol. 20, Issue 12, 2900-2908, June 15, 2009

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Varp Is a Novel Rab32/38-binding Protein That Regulates Tyrp1 Trafficking in Melanocytes

Kanako Tamura^*,, Norihiko Ohbayashi^*,, Yuto Maruta^*, Eiko Kanno^*, Takashi Itoh^*, and Mitsunori Fukuda^*

*Laboratory of Membrane Trafficking Mechanisms, Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai, Miyagi 980-8578, Japan

Submitted December 2, 2008; Revised April 15, 2009; Accepted April 16, 2009
Monitoring Editor: Thomas F.J. Martin

Two small GTPase Rabs, Rab32 and Rab38, have recently been proposed to regulate trafficking of melanogenic enzymes to melanosomes in mammalian epidermal melanocytes; however, the exact molecular mechanism of Rab32/38-mediated transport of melanogenic enzymes has never been clarified, because no Rab32/38-specific effector has ever been identified. In this study, we screened for a Rab32/38-specific effector by a yeast two-hybrid assay using a guanosine triphosphate (GTP)-locked Rab32/38 as bait and found that VPS9-ankyrin-repeat protein (Varp)/Ankrd27, characterized previously as a guanine nucleotide exchange factor (GEF) for Rab21, functions as a specific Rab32/38-binding protein in mouse melanocyte cell line melan-a. Deletion analysis showed that the first ankyrin-repeat (ANKR1) domain functions as a GTP-dependent Rab32/38-binding domain, but that the N-terminal VPS9 domain (i.e., Rab21-GEF domain) does not. Small interfering RNA-mediated knockdown of endogenous Varp in melan-a cells caused a dramatic reduction in Tyrp1 (tyrosinase-related protein 1) signals from melanosomes but did not cause any reduction in Pmel17 signals. Furthermore, expression of the ANKR1 domain in melan-a cells also caused a dramatic reduction of Tyrp1 signals, whereas the VPS9 domain had no effect. Based on these findings, we propose that Varp functions as the Rab32/38 effector that controls trafficking of Tyrp1 in melanocytes.

These authors are equally contributed to this work.

Address correspondence to: Mitsunori Fukuda (nori{at}mail.tains.tohoku.ac.jp)

Abbreviations used: ANKR, ankyrin-repeat; GEF, guanine nucleotide exchange factor; GFP, green fluorescent protein; GST, glutathione transferase; HRP, horseradish peroxidase; shRNA, short hairpin RNA; siRNA, small interfering RNA; Tyrp1, tyrosinase-related protein 1; Str, monomeric strawberry; Varp, VPS9-ankyrin-repeat protein; VPS9, vacuolar protein sorting 9.

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