Technical Reference #1602

1602.

Varp is a novel Rab32/38 protein that regulates Tyrp1 trafficking in melanocytes Kanako Tamura; Norihiko Ohbayashi; Yuto Maruta; Eiko Kanno; Takashi Itoh; Mitsunori Fukuda, Tohoku University, Molecular Biology of the Cell, 20(1602), (2009)
Link To Paper

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 enzy

Keywords:
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Materials & Methods:
Plasmid construction. pEF-T7-Slac2-a or pEGFP-C1-Slac2-a deletion mutants were constructed essentially by conventional PCR as described previously (11 13 15 17). The SHD includes amino acid residues 1 to 153 of mouse Slac2-a the MBD includes amino acid residues 241 to 405 of mouse Slac2-a the ABD includes amino acid residues 401 to 590 of mouse Slac2-a; and ABD includes amino acid residues 1 to 480 of mouse Slac2-a. Other expression constructs (pEF-FLAG-MC-myosin Va-tail pEF-FLAG-Rab27A pEF-HA-Rab27A and pEGFP-C1-Slac2-a) were prepared as described previously (13 27). Mutant Slac2-a plasmids carrying an EA (D378A/E380A/E381A/E382A) or KA (K493A/R495A/R496A/K497A) substitution were produced by two-step PCR techniques with the following mutagenic oligonucleotides with an artificial SacII or SphI site (underlined) respectively as described previously (14): 5'-CCGCGGCTGTGGCAGCACTGGG-3' (EA primer 1; antisense) 5'-GCCGCGGCGACACTCAGGAGGA-3' (EA primer 2; sense) 5'-GGCATGCCTGACGCTGCCGCAGGTGCTCCCGAGGGCTTCACTGTGAG-3' (KA primer 1; antisense) and 5'-GGCATGCCGATCTTTCTTCC-3' (KA primer 2; sense). The mutant Slac2-a fragments were subcloned into the pEF-T7 tag vector (11 15) with appropriate restriction enzyme sites. Cell culture and transfection. COS-7 cells were cultured in Dulbecco's modified Eagle's medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum 100 U of penicillin G per ml and 100 µg of streptomycin per ml at 37°C under 5% CO2. Lipofectamine Plus (Invitrogen) was used for transfections of plasmids into COS-7 cells as described previously (17). The immortal mouse melanocyte cell line melan-a derived from a black mouse (3) was kindly provided by Dorothy C. Bennett (St. George's Hospital Medical School London United Kingdom) and Katsuhiko Tsukamoto (University of Yamanashi Yamanashi Japan). Melan-a cells were cultured in RPMI 1640 medium (Sigma-Aldrich) supplemented with 2.7 mM HCl 10% fetal bovine serum 100 U of penicillin G per ml 100 µg of streptomycin per ml 7.5 µg of phenol red per ml and 0.1 mM 2-mercaptoethanol at 37°C under 10% CO2. Immediately prior to use 200 nM phorbol 12-myristate 13-acetate (Sigma-Aldrich) was added to the medium. For subculture melan-a cells were treated with 250 µg of trypsin per ml-0.1 mM EDTA. Fugene 6 (Roche) was used for transfection of pEGFP-C1 plasmids into melan-a cells according to the manufacturer's instructions. Immunoprecipitation. T7-tagged Slac2-a FLAG-tagged MC-myosin Va-tail and/or hemagglutinin (HA)-tagged Rab27A was coexpressed in COS-7 cells and recombinant proteins were solubilized at 4°C for 1 h with a buffer containing 1% Triton X-100 250 mM NaCl 1mM MgCl2 50 mM HEPES-KOH (pH 7.2) and appropriate protein inhibitors. T7-Slac2-a was immunoprecipitated with anti-T7 tag antibody-conjugated agarose (Novagen) as described previously (11 15). Coimmunoprecipitated FLAG-myosin Va-tail HA-Rab27A and actin were first detected with horseradish peroxidase-conjugated anti-FLAG tag antibody (Sigma-Aldrich) anti-HA tag antibody (Roche) and antiactin antibody (Santa Cruz Biotechnology) respectively and the immunoprecipitated T7-Slac2-a proteins were then detected with horseradish peroxidase-conjugated anti-T7 tag antibody (Novagen) as described previously (7 15 18). Immunoreactive bands were visualized by enhanced chemiluminescence (Amersham Biosciences). The blots shown in this paper are representative of those from at least two or three independent experiments. Immunofluorescence and melanosome distribution assay. Melan-a cells (105 cells [the day before transfection] per 35-mm-diameter glass-bottom dish; MatTek Corp.) were transfected with 2 µg of a plasmid encoding a green fluorescent protein (GFP)-tagged Slac2-a protein with 3 µl of Fugene 6. At 36 to 48 h after transfection cells were fixed with 4% paraformaldehyde (catalog no. 168-20955; Wako Pure Chemicals) for 20 min and permeabilized with 0.3% Triton X-100 for 2 min. The cells were then immunostained with anti-Rab27A mouse immunoglobulin G (IgG) (1/50 dilution; BD Transduction Laboratories) and anti-myosin Va rabbit IgG (6.9 µg/ml) (18) followed by anti-rabbit Alexa Fluor 633 IgG and anti-mouse Alexa Fluor 568 IgG (1/5000 dilution; Molecular Probes) as described previously (10 27). Images were acquired and pseudocolored with a confocal laser scan microscope (Fluoview; Olympus) and processed with Adobe Photoshop software (version 7.0). Outlines of cells were depicted by Fluoview. Melanosome distribution was assayed by examination of images of transfected melan-a cells (more than 50 cells/dish three independent dishes for each plasmid) obtained at random. Cells in which more than 50% of melanosomes were present around the nucleus were judged to be aggregated. The absence of melanosomes in the periphery (a phenotype observed in GFP-ABD-transfected cells) was distinguished from perinuclear aggregation of melanosomes by the loss of large clumps of melanosomes around the nucleus.

Microscopic Technique
Confocal Microscopy, Fluorescence Microscopy, Light Microscopy

Cell Type(s)
melan-a