title: Assembly of the biogenesis of lysosome-related organelles complex-3 (BLOC-3) and its interaction with Rab9.
abstract: 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.
authors.affiliation: Laboratory of Molecular Biology, NIDDK, Eunice Kennedy Shriver NICHD, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Daniel P
authors.initials: DP
authors.lastname: Kloer
authors.forename: Raul
authors.initials: R
authors.lastname: Rojas
authors.forename: Viorica
authors.initials: V
authors.lastname: Ivan
authors.forename: Kengo
authors.initials: K
authors.lastname: Moriyama
authors.forename: Thijs
authors.initials: T
authors.lastname: van Vlijmen
authors.forename: Namita
authors.initials: N
authors.lastname: Murthy
authors.forename: Rodolfo
authors.initials: R
authors.lastname: Ghirlando
authors.forename: Peter
authors.initials: P
authors.lastname: van der Sluijs
authors.forename: James H
authors.initials: JH
authors.lastname: Hurley
authors.forename: Juan S
authors.initials: JS
authors.lastname: Bonifacino
mesh_terms: D000818:Animals; D001665:Binding Sites; D020662:Guanine Nucleotide Exchange Factors; D006160:Guanosine Triphosphate / Q000378:metabolism; D022861:Hermanski-Pudlak Syndrome / Q000235:genetics / Q000473:pathology / Q000503:physiopathology; D006801:Humans; D008247:Lysosomes / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism*; D015388:Organelles / Q000378:metabolism*; D011479:Protamines / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D020691:rab GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D020662:Guanine Nucleotide Exchange Factors; C451752:HPS4 protein, human; D046912:Multiprotein Complexes; D011479:Protamines; D021122:Protein Subunits; D011506:Proteins; D011993:Recombinant Fusion Proteins; C068621:protamine 2; D006160:Guanosine Triphosphate; C079856:RAB9A protein, human; D020691:rab GTP-Binding Proteins