Structure-Function Analysis of VPS9-Ankyrin-repeat Protein (Varp) in the Trafficking of Tyrosinase-related Protein 1 in Melanocytes

Abstract

Because Varp (VPS9-ankyrin-repeat protein)/Ankrd27 specifically binds two small GTPases, Rab32 and Rab38, which redundantly regulate the trafficking of melanogenic enzymes in mammalian epidermal melanocytes, it has recently been implicated in the regulation of trafficking of a melanogenic enzyme tyrosinase-related protein 1 (Tyrp1) to melanosomes. However, the functional interaction between Rab32/38 and Varp and the involvement of the VPS9 domain (i.e. Rab21-GEF domain) in Tyrp1 trafficking have never been elucidated. In this study, we succeeded in identifying critical residues of Rab32/38 and Varp that are critical for the formation of the Rab32/38⅐Varp complex by performing Ala-based site-directed mutagenesis, and we dis- Pigmentation of mammalian hair and skin requires proper formation and transport of melanosomes, one of the lysosomerelated organelles that specifically synthesize and store melanin pigments, in melanocytes (reviewed in Refs. 1, 2). Defects in the formation and/or transport of melanosomes often cause pigmentary disorders, e.g. albinism and pigmentary dilution, in mammals (reviewed in Ref.3). The formation and transport of melanosomes involve a variety of intracellular membrane trafficking events, and several distinct Rab-type small GTPases, which are conserved membrane trafficking proteins in all eukaryotic cells (reviewed in Refs. 4 -6), have been shown to regulate the maturation and transport of melanosomes in mammalian epidermal melanocytes.The best characterized Rab isoform that is abundant on mature melanosomes in melanocytes is Rab27A (7-9). Rab27A regulates actin-based melanosome transport through interaction with its specific effector, Slac2-a/melanophilin (10 -12), and melanosome anchoring to the plasma membrane through interaction with another effector, Slp2-a (13). As a result, Rab27A deficiency causes human type 2 Griscelli syndrome, which is characterized by silvery hair (i.e. partial albinism) (reviewed in Ref. 14 and references therein). Two other Rab isoforms, Rab32 and Rab38, regulate an early step in melanogenesis, i.e. the transport of melanogenic enzymes to melanosomes (15-17). Actually, dysfunction of Rab38 causes the diluted coat color of chocolate mice, presumably because of impairment of the targeting of tyrosinase-related protein 1 (Tyrp1) to melanosomes (15). The discovery of the Rab32/38-specific binding protein Varp 4 (VPS9-ankyrin-repeat protein; official symbol in the National Center for Biotechnology Information is Ankrd27) has recently been reported (18,19), and as its name indicates, Varp protein contains an N-terminal VPS9 (vacuolar protein sorting 9) domain and C-terminal tandem ankyrin repeat domains (named ANKR1 and ANKR2). The Varp VPS9 domain possesses Rab21-GEF (guanine nucleotide exchange factor) activity (20), and the ANKR1 domain functions as a specific GTP-Rab32/38-binding site (18,19). Even more recently, VAMP7/TI-VAMP (vesicle-associated membrane protein) has been shown to interact with the region between the ANKR1 domain and the ANKR2 domain and t...

RUTBC1 Functions as a GTPase-activating Protein for Rab32/38 and Regulates Melanogenic Enzyme Trafficking in Melanocytes

“…5B). Consistent with these results, the knockdown of Varp, a Rab32/38 effector (14,15), in melan-a cells also resulted in a reduction of Dct signals as well as tyrosinase/Tyrp1 signals according to both the immunostaining (Fig. 5C) and the immunoblotting (Fig.…”

“…The siRNA against mouse Rab32 has been described previously (15 RT-PCR-The total RNA of melan-a cells transfected with RUTBC1 siRNA and control siRNA were prepared with TRI Reagent (Sigma-Aldrich), and reverse transcription was performed by using ReverTra Ace (Toyobo, Osaka, Japan) according to the manufacturer's instructions. The following pairs of oligonucleotides were used for amplification: for mouse RUTBC1, forward primer, 5Ј-GCATCCAGTCCAGCCTAGAT-3Ј, and reverse primer, 5Ј-GCAGGAACCAGCGATAACAG-3Ј; and for GAPDH, forward primer, 5Ј-ATGGTGAAGGTCG-GAGTCAA-3Ј, and reverse primer, 5Ј-GCCATGTAGAC-CATGAGGTC-3Ј.…”

“…RUTBC1 Possesses GAP Activity toward Both Rab32 and Rab38 -The inhibitory effect of the RUTBC1 overexpression on the trafficking of all three melanogenic enzymes led us to hypothesize that in living cells RUTBC1 possesses GAP activity toward Rab32 and Rab38, both of which redundantly regulate the trafficking of tyrosinase and Tyrp1 (3,11,14,15). To test our hypothesis, we investigated the endogenous Rab32/38 expression in T7-RUTBC1-C-expressing cells by performing immunostaining.…”

“…Because activation of Rab32/38 is essential for the trafficking of tyrosinase/Tyrp1/Dct (3,11,12,15) (Fig. 5), we initially thought that RUTBC1 knockdown, i.e.…”

“…on the melanosomal localization of either of these melanogenic enzymes (3,11,15). Because a Tre-2/Bub2/Cdc16 (TBC) domain-containing protein, RUTBC1, has been shown to possess GAP activity toward Rab32 but with roughly 10-fold lower activity toward Rab38 in vitro (18), overexpression of RUTBC1 in melanocytes was expected to have no or little effect on the expression of tyrosinase and Tyrp1, the same as in Rab32 single knockdown cells (3,11).…”