QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 27 November 2007
doi: 10.1242/jcs.017913

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Kobayashi, T. Articles by Hearing, V. J. Search for Related Content PubMed PubMed Citation Articles by Kobayashi, T. Articles by Hearing, V. J.

Direct interaction of tyrosinase with Tyrp1 to form heterodimeric complexes in vivo

¹ Department of Physiology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
² Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA

View larger version (46K): [in this window] [in a new window]   Fig. 1. Specificity of guinea pig antibodies for melanosomal proteins. (A) Analysis of antibody reactivities by immunopurification, immunodepletion and western blot analysis of melanocyte extracts. Tyr, Tyrp1, Dct or Silver proteins were immunopurified (left) from an extract of melan-a melanocytes using antibodies as noted at the bottom and then were analyzed by western blot using antibodies as noted on the left. Subscripts of `GP' or `R' refer to antisera generated in guinea pigs or rabbits, respectively; NS, normal serum. An extract of melan-a melanocytes was immunodepleted with antibody and protein-G-Sepharose (middle). Reactivity could be blocked by pre-incubation with the corresponding antigenic peptide (data not shown). Protein extracts from melan-a, melan-b or melan-c melanocytes were separated by SDS-PAGE (right). (B) Intracellular distribution of Tyr and Tyrp1 detected immunohistochemically. Melan-a melanocytes were fixed in 4% paraformaldehyde and stained for Tyr and Tyrp1. Tyrp1-positive (red) vesicles that colocalized with Tyr (green) are seen as yellow in the merged image. (Note that a relatively small fraction of vesicles is yellow, which may reflect cell variability in gene expression or antigen masking in melanized melanosomes.) Bar, 10 µm.

View larger version (46K): [in this window] [in a new window]   Fig. 2. Interaction of Tyr and Tyrp1 in melanocytes. Melan-a melanocytes were incubated in ice-cold PBS containing 0, 0.5 or 1 mM DSP (as indicated) for 15 minutes on ice, followed by quenching with 2 mM glycine/PBS and exhaustive washing with PBS. After solubilization, equal amounts of each cell extract were immunoprecipitated and melanogenic proteins were analyzed by SDS-PAGE and immunoblotting. Immunoprecipitated and coprecipitated proteins were separated by SDS-PAGE under reducing conditions, which cleaves the crosslinks, and proteins migrate at their original molecular sizes. Subscripts of `GP' or `R' refer to antisera generated in guinea pigs or rabbits, respectively; NS, normal serum.

View larger version (41K): [in this window] [in a new window]   Fig. 3. Interaction of Tyr and Tyrp1 in melanosomes. Melanosomes were purified from melan-a melanocytes. Interaction of Tyr and Tyrp1 was analyzed by chemical crosslinking, immunoprecipitation and immunoblotting, as described for Fig. 2.

View larger version (53K): [in this window] [in a new window]   Fig. 4. Interaction of Tyr and Tyrp1 in cycloheximide-treated melanocytes. (A) Confocal images of melan-a melanocytes treated with or without cycloheximide. Melanocytes expressing YFP-ER or YFP-Golgi were treated with cycloheximide (10 µg/ml) for 10 hours, fixed and stained with the TyrGP antibody as described for Fig. 1. Melanocytes treated with cycloheximide were also double-stained with TyrR, Tyrp1GP or HMB45 as noted. Bright-field images are shown to view pigment granules in the cells. Bar, 10 µm. (B) Interaction of Tyr and Tyrp1 in cycloheximide-treated melan-a melanocytes was analyzed by chemical crosslinking with DSP, immunoprecipitation and immunoblotting, as described for Fig. 2.

View larger version (24K): [in this window] [in a new window]   Fig. 5. Direct interaction of Tyr and Tyrp1. Tyr was immunoprecipitated from melan-a melanocytes pretreated with or without DSP, and was separated in the first dimension by SDS-PAGE under nonreducing conditions. Tyr and crosslinked proteins were cleaved and resolved in the second dimension by SDS-PAGE under reducing conditions. Blots of DSP-treated samples detected by anti-TyrGP (green) and Tyrp1 GP (red) were overlaid and are shown in the bottom panel. Arrowheads indicate Tyr or Tyrp1 detectable only when cells had been pretreated with DSP. Tyr spots marked with arrows were detected regardless of DSP treatment and probably represent Tyr that comigrated with the anti-Tyr IgG heavy chain on the first gel. There are also some non-specific spots visible at 58 kDa. Representative results of two experiments are shown.

View larger version (30K): [in this window] [in a new window]   Fig. 6. Interaction of Tyr or Tyrp1 with other melanosomal proteins. Tyr or Tyrp1 was immunoprecipitated from melan-a melanocytes pretreated with or without 1 mM DSP as described for Fig. 2. Immunoblotting analysis was used to detect Tyr/Tyrp1 coprecipitates using TyrGP, Tyrp1GP, DctGP and SilverGP antibodies.

View larger version (49K): [in this window] [in a new window]   Fig. 7. Interaction of Tyr with Tyrp1 is disrupted in Tyrp1b mutant melanocytes. Tyr and Tyrp1 were immunoprecipitated from melan-b melanocytes by antibodies as noted at the bottom and then were examined by western blot analysis as detailed in Fig 2.