The BLOC-3 subunit HPS4 is required for activation of Rab32/38 GTPases in melanogenesis, but its Rab9 activity is dispensable for melanogenesis

J Biol Chem. 2019 Apr 26;294(17):6912-6922. doi: 10.1074/jbc.RA119.007345. Epub 2019 Mar 5.

Abstract

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.

Keywords: BLOC-3; Hermansky–Pudlak syndrome; Rab; Rab9; guanine nucleotide exchange factor (GEF); hypopigmentation; melanogenesis; melanosome; membrane trafficking; tyrosinase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • COS Cells
  • Cell Line, Transformed
  • Chlorocebus aethiops
  • Enzyme Activation
  • Guanine Nucleotide Exchange Factors / chemistry
  • Guanine Nucleotide Exchange Factors / physiology*
  • Melanins / biosynthesis*
  • Mice
  • Mutagenesis, Site-Directed
  • Protein Binding
  • rab GTP-Binding Proteins / metabolism*

Substances

  • Guanine Nucleotide Exchange Factors
  • Hps4 protein, mouse
  • Melanins
  • Rab32 protein, mouse
  • Rab38 protein, mouse
  • rab9 protein, mouse
  • rab GTP-Binding Proteins