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

Abstract

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.

Structure-Function Analyses of the Small GTPase Rab35 and Its Effector Protein Centaurin-β2/ACAP2 during Neurite Outgrowth of PC12 Cells

“…We previously thought that the ANKR domain of centaurin-␤2 was necessary and sufficient for Rab35 binding activity, the same as the ANKR1 domain of VPS9-ankyrin repeat protein that alone recognizes Rab32/38 (32), but the results of our deletion analysis indicated that an N-terminal flanking region and a C-terminal flanking region (AA580 -630 and AA730 -755, respectively) of the ANKR domain are also required for high affinity Rab35 binding activity (Fig. 5).…”

“…Yeast Two-hybrid Assays-Yeast two-hybrid assays were performed by using pGBD-C1-Rab35(Q67L, Q67L/T76S/T81A, or Q67L/S5A) lacking the C-terminal geranylgeranylation site and pGAD-C1-RUSC2-RUN (12), pAct2-MICAL-1, pAct2-MICAL-cl, pAct2-MICAL-L1, pAct2-OCRL (7), pAct2-Fascin1, or pAct2-Cent␤2-ANKR (8) or by using pGBD-C1-Rab35(Q67L or S22N)⌬Cys (hereafter simply designated as QL or SN) and pAct2-Cent␤2-ANKR(WT or its mutants), pAct2-Cent␤1-ANKR, or pAct2-Cent␤5-ANKR as described previously (8,32). The yeast strain, medium, culture conditions, and transformation protocol used were as described previously (28).…”

“…In an attempt to identify the structural determinant responsible for the exclusive Rab35 binding specificity of centaurin-␤2, we first focused on the switch II region of Rabs that interact with most of the Rab35BPs (Fig. 1A) because several amino acids in the switch II region of certain Rabs have previously been shown to be responsible for specific effector binding (32)(33)(34)(35)(36)(37)(38). Because Rab1A/B, Rab8A/B, Rab10, Rab13, Rab15, and Rab35 are phylogenetically similar and belong to the same large branch in the phylogenetic tree (39), the amino acid sequences of their switch II region are highly conserved.…”

“…Rab7-binding site) (40) and the ANKR domain of VPS9-ankyrin repeat protein (Varp) (i.e. Rab32/38-binding site) (32,41) with the centaurin-␤2 ANKR domain. Because of their low sequence conservation, however, we were unable to identify shared key residues that are responsible for Rab binding by these three ANKR domains.…”

Rabex-5 Protein Regulates Dendritic Localization of Small GTPase Rab17 and Neurite Morphogenesis in Hippocampal Neurons

“…Rabex-5 to dendrite outgrowth and axon morphogenesis and Rab17 to dendrite morphogenesis) suggests that Rabex-5 activates other Rab family proteins besides Rab17 during the course of neurite morphogenesis. To pursue this possibility, we performed yeast two-hybrid assays with a panel of 60 different CA (GTP-locked) and CN (GDP-locked) Rabs to identify additional target Rabs of Rabex-5 (36). The results of the Rab-family-wide analysis showed that Rabex-5 specifically interacted with the CN forms of Rab5A/B/C, Rab17, and Rab21 (Fig.…”