TY - JOUR
T1 - The BLOC-3 subunit HPS4 is required for activation of Rab32/38 GTPases in melanogenesis, but its Rab9 activity is dispensable for melanogenesis
AU - Ohishi, Yuta
AU - Kinoshita, Riko
AU - Marubashi, Soujiro
AU - Ishida, Morié
AU - Fukuda, Mitsunori
N1 - Funding Information:
1 Supported by the Japan Society for the Promotion of Science.
Funding Information:
This work was supported in part by Grant-in-Aid for Scientific Research (B) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan 15H04367 (to M. F.), Grant-in-Aid for Scientific Research on Innovative Areas from MEXT 17H05682 (to M. F.), Japan Science and Tech-nology Agency (JST) CREST Grant JPMJCR17H4 (to M. F.), a grant from the Hoyu Science Foundation (to M. F.), and a grant from the Kao Melanin Workshop (to S. M.). The authors declare that they have no conflicts of interest with the contents of this article.
Publisher Copyright:
© 2019 Ohishi et al.
PY - 2019/4/26
Y1 - 2019/4/26
N2 - HPS4 biogenesis of lysosome-related organelles complex 3 subunit 2 (HPS4) is one of the genes whose mutations have been associated with Hermansky-Pudlak syndrome (HPS), characterized by ocular albinism and susceptibility to bleeding because of defects in the biogenesis of lysosome-related organelles such as melanosomes. HPS4 protein forms a BLOC-3 complex with HPS1, another HPS gene product, and the complex has been proposed to function as a guanine nucleotide exchange factor (GEF) for RAB32, a member of the Rab small GTPase family (Rab32), and Rab38 (Rab32/38-GEF) and also as a Rab9 effector. Although both Rab32/38 and Rab9 have been shown previously to be involved in melanogenesis in mammalian epidermal melanocytes, the functional relationships of these small GTPases with BLOC-3 remain unknown. In this study, we used site-directed mutagenesis to generate HPS4 mutants that specifically lack either Rab32/38-GEF activity or Rab9- binding activity and investigated their involvement in melanogenesis of melan-le cells (an HPS4-deficient melanocyte cell line derived from light ear mice). Melan-le cells exhibit a clear hypopigmentation phenotype, i.e. reduced expression and abnormal distribution of tyrosinase and reduced melanin content. Although re-expression of WT HPS4 completely rescued this phenotype, the Rab32/38-GEF activity- deficient HPS4 mutant failed to restore melanin content and tyrosinase trafficking in these cells. Unexpectedly, as WT HPS4, the Rab9 binding-deficient HPS4 mutant completely rescued the phenotype. These results indicate that activation of Rab32/38 by HPS4 (or BLOC-3) is essential for melanogenesis of cultured melanocytes and that Rab9 likely regulates melanogenesis independently of HPS4.
AB - HPS4 biogenesis of lysosome-related organelles complex 3 subunit 2 (HPS4) is one of the genes whose mutations have been associated with Hermansky-Pudlak syndrome (HPS), characterized by ocular albinism and susceptibility to bleeding because of defects in the biogenesis of lysosome-related organelles such as melanosomes. HPS4 protein forms a BLOC-3 complex with HPS1, another HPS gene product, and the complex has been proposed to function as a guanine nucleotide exchange factor (GEF) for RAB32, a member of the Rab small GTPase family (Rab32), and Rab38 (Rab32/38-GEF) and also as a Rab9 effector. Although both Rab32/38 and Rab9 have been shown previously to be involved in melanogenesis in mammalian epidermal melanocytes, the functional relationships of these small GTPases with BLOC-3 remain unknown. In this study, we used site-directed mutagenesis to generate HPS4 mutants that specifically lack either Rab32/38-GEF activity or Rab9- binding activity and investigated their involvement in melanogenesis of melan-le cells (an HPS4-deficient melanocyte cell line derived from light ear mice). Melan-le cells exhibit a clear hypopigmentation phenotype, i.e. reduced expression and abnormal distribution of tyrosinase and reduced melanin content. Although re-expression of WT HPS4 completely rescued this phenotype, the Rab32/38-GEF activity- deficient HPS4 mutant failed to restore melanin content and tyrosinase trafficking in these cells. Unexpectedly, as WT HPS4, the Rab9 binding-deficient HPS4 mutant completely rescued the phenotype. These results indicate that activation of Rab32/38 by HPS4 (or BLOC-3) is essential for melanogenesis of cultured melanocytes and that Rab9 likely regulates melanogenesis independently of HPS4.
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U2 - 10.1074/jbc.RA119.007345
DO - 10.1074/jbc.RA119.007345
M3 - Article
C2 - 30837268
AN - SCOPUS:85065089967
VL - 294
SP - 6912
EP - 6922
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 17
ER -