guanosine-triphosphate and Hermanski-Pudlak-Syndrome

The Hermansky-Pudlak syndrome (HPS) is a genetic hypopigmentation and bleeding disorder caused by defective biogenesis of lysosome-related organelles (LROs) such as melanosomes and platelet dense bodies. HPS arises from mutations in any of 8 genes in humans and 16 genes in mice. Two of these genes, HPS1 and HPS4, encode components of the biogenesis of lysosome-related organelles complex-3 (BLOC-3). Herein we show that recombinant HPS1-HPS4 produced in insect cells can be efficiently isolated as a 1:1 heterodimer. Analytical ultracentrifugation reveals that this complex has a molecular mass of 146 kDa, equivalent to that of the native complex and to the sum of the predicted molecular masses of HPS1 and HPS4. This indicates that HPS1 and HPS4 interact directly in the absence of any other protein as part of BLOC-3. Limited proteolysis and deletion analyses show that both subunits interact with one another throughout most of their lengths with the sole exception of a long, unstructured loop in the central part of HPS4. An interaction screen reveals a specific and strong interaction of BLOC-3 with the GTP-bound form of the endosomal GTPase, Rab9. This interaction is mediated by HPS4 and the switch I and II regions of Rab9. These characteristics indicate that BLOC-3 might function as a Rab9 effector in the biogenesis of LROs.

Topics: Animals; Binding Sites; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate; Hermanski-Pudlak Syndrome; Humans; Lysosomes; Mice; Models, Molecular; Multiprotein Complexes; Organelles; Protamines; Protein Binding; Protein Multimerization; Protein Structure, Tertiary; Protein Subunits; Proteins; rab GTP-Binding Proteins; Recombinant Fusion Proteins; Two-Hybrid System Techniques

Rab38 is a low-molecular-weight G-protein highly expressed in melanocytes of the skin and alveolar type II cells in the lung. A point mutation in the postulated GTP/GDP-interacting domain of Rab38 has been identified as the genetic lesion responsible for oculocutaneous albinism (OCA) in chocolate (cht) mice. Another point mutation that prevents translation of Rab38 mRNA is the molecular basis of the Ruby gene mutation causing the phenotype of OCA and prolonged bleeding time in Fawn-Hooded and Tester-Moriyama rats. Cht mice show conspicuously enlarged lamellar bodies in alveolar type II cells and abnormal lung structure. Triton X-114 phase partitioning of cht mouse lung showed that Rab38cht-protein was recovered in the aqueous phase. We produced recombinant Rab38cht-protein using a baculovirus/insect cell-protein expression system. The results demonstrate that Rab38cht-protein is inactive due to reduced membrane binding and enhanced intracellular degradation. Rab38 is a new strong candidate gene for human Hermansky-Pudlak syndrome (HPS) that is characterized by OCA, bleeding diathesis, and lung disease.

Topics: Albinism, Oculocutaneous; Animals; Female; Guanosine Triphosphate; Hermanski-Pudlak Syndrome; Humans; Lung Diseases; Male; Mice; Microscopy, Electron; rab GTP-Binding Proteins; Rats; Recombinant Proteins