Hermansky-Pudlak Syndrome Protein Complexes Associate with Phosphatidylinositol 4-Kinase Type II �� in Neuronal and Non-neuronal Cells

The Hermansky-Pudlak syndrome is a disorder affecting endosome sorting.Disease is triggered by defects in any of 15 mouse gene products, which arepart of five distinct cytosolic molecular complexes: AP-3, homotypic fusionand vacuole protein sorting, and BLOC-1, -2, and -3. To identify molecularassociations of these complexes, we used in vivo cross-linkingfollowed by purification of cross-linked AP-3 complexes and mass spectrometricidentification of associated proteins. AP-3 was co-isolated with BLOC-1,BLOC-2, and homotypic fusion and vacuole protein sorting complex subunits;clathrin; and phosphatidylinositol-4-kinase type II α (PI4KIIα).We previously reported that this membrane-anchored enzyme is a regulator ofAP-3 recruitment to membranes and a cargo of AP-3 (Craige, B.,Salazar, G., and Faundez, V. (2008) Mol. Biol.Cell19,1415-1426). Using cells deficientin different Hermansky-Pudlak syndrome complexes, we identified that BLOC-1,but not BLOC-2 or BLOC-3, deficiencies affect PI4KIIα inclusion intoAP-3 complexes. BLOC-1, PI4KIIα, and AP-3 belong to a tripartitecomplex, and down-regulation of either PI4KIIα, BLOC-1, or AP-3complexes led to similar LAMP1 phenotypes. Our analysis indicates that BLOC-1complex modulates the association of PI4KIIα with AP-3. These resultssuggest that AP-3 and BLOC-1 act, either in concert or sequentially, tospecify sorting of PI4KIIα along the endocytic route.Membranous organelles along the exocytic and endocytic pathways are eachdefined by unique lipid and protein composition. Vesicle carriers communicateand maintain the composition of these organelles(2). Consequently defining themachineries that specify vesicle formation, composition, and delivery arecentral to understanding membrane protein traffic. Generally vesiclebiogenesis uses multiprotein cytosolic machineries to select membranecomponents for inclusion in nascent vesicles(2,3). Heterotetrameric adaptorcomplexes (AP-1 to AP-4) are critical to generate vesicles of specificcomposition from the different organelles constituting the exocytic andendocytic routes(2-4).The best understood vesicle formation machinery in mammalian cells is theone organized around the adaptor complex AP-2(5). This complex generatesvesicles from the plasma membrane using clathrin. Our present detailedunderstanding of AP-2 vesicle biogenesis mechanisms and interactions emergedfrom a combination of organellar and in vitro binding proteomicsanalyses together with the study of binary interactions in cell-free systems(5-9).In contrast, the vesicle biogenesis pathways controlled by AP-3 are far lessunderstood. AP-3 functions to produce vesicles that traffic selected membraneproteins from endosomes to lysosomes, lysosome-related organelles, or synapticvesicles(10-13).AP-3 is one of the protein complexes affected in the Hermansky-Pudlak syndrome(HPS;³ OnlineMendelian Inheritance in Man (OMIM) 203300). So far, mutations in any of 15mouse or eight human genes trigger a common syndrome. This syndromeencompasses defects that include pigment dilution, platelet dysfunction,pulmonary fibrosis, and occasionally neurological phenotypes(14,15). All forms of HPS showdefective vesicular biogenesis or trafficking that affects lysosomes,lysosome-related organelles (for example melanosomes and platelet densegranules), and, in some of them, synaptic vesicles(11-13).Most of the 15 HPS loci encode polypeptides that assemble into five distinctmolecular complexes: the adaptor complex AP-3, HOPS, and the BLOC complexes 1,2, and 3 (14). Recently binaryinteractions between AP-3 and BLOC-1 or BLOC-1 and BLOC-2 suggested thatarrangements of these complexes could regulate membrane protein targeting(16). Despite the abundance ofgenetic deficiencies leading to HPS and genetic evidence that HPS complexesmay act on the same pathway in defined cell types(17), we have only a partialpicture of protein interactions organizing these complexes and how they mightcontrol membrane protein targeting.In this study, we took advantage of cell-permeant and reversiblecross-linking of HPS complexes followed by their immunoaffinity purificationto identify novel molecular interactions. Cross-linked AP-3 co-purified withBLOC-1, BLOC-2, HOPS, clathrin, and the membrane protein PI4KIIα. Wepreviously identified PI4KIIα as a cargo and regulator of AP-3recruitment to endosomes (1,18). Using mutant cellsdeficient in either individual HPS complexes or a combination of them, wefound that BLOC-1 facilitates the interaction of AP-3 and PI4KIIα. Ourstudies demonstrate that subunits of four of the five HPS complexes co-isolatewith AP-3. Moreover BLOC-1, PI4KIIα, and AP-3 form a tripartite complexas demonstrated by sequential co-immunoprecipitations as well as by similarLAMP1 distribution phenotypes induced by down-regulation of components of thistripartite complex. Our findings indicate that BLOC-1 complex modulates therecognition of PI4KIIα by AP-3. These data suggest that AP-3, either inconcert or sequentially with BLOC-1, participates in the sorting of commonmembrane proteins along the endocytic route.     

Plasmodium falciparum UvrD Helicase Translocates in 3′ to 5′ Direction,Colocalizes with MLH and Modulates Its Activity through Physical Interaction

Malaria is a global disease and a major health problem. The control of malaria is a daunting task due to the increasing drug resistance. Therefore, there is an urgent need to identify and characterize novel parasite specific drug targets. In the present study we report the biochemical characterization of parasite specific UvrD helicase from Plasmodium falciparum. The N-terminal fragment (PfUDN) containing UvrD helicase domain, which consists of helicase motifs Q, Ia–Id, II, III and most of motif IV, and the C-terminal fragment (PfUDC1) containing UvrD helicase C terminal domain, consisting of remaining part of motif IV and motifs IVa–IVc and 161 amino acids of intervening sequence between motif IV and V, possess ssDNA-dependent ATPase and DNA helicase activities in vitro. Using immunodepletion assays we show that the ATPase and helicase activities are attributable to PfUDN and PfUDC1 proteins. The helicase activity can utilize the hydrolysis of all the nucleotide and deoxynucleotide triphosphates and the direction of unwinding is 3′ to 5′. The endogenous P. falciparum UvrD contains the characteristic DNA helicase activity. PfUDN interacts with PfMLH (P. falciparum MutL homologue) and modulates the endonuclease activity of PfMLH and PfMLH positively regulates the unwinding activity of PfUDN. We show that PfUvrD is expressed in the nucleus distinctly in the schizont stages of the intraerythrocytic development of the parasite and it colocalizes with PfMLH. These studies will make an important contribution in understanding the nucleic acid transaction in the malaria parasite.