Abstract
The Hermansky-Pudlak syndrome is a disorder affecting endosome sorting. Disease is triggered by defects in any of 15 mouse gene products, which are part of five distinct cytosolic molecular complexes: AP-3, homotypic fusion and vacuole protein sorting, and BLOC-1, -2, and -3. To identify molecular associations of these complexes, we used in vivo cross-linking followed by purification of cross-linked AP-3 complexes and mass spectrometric identification 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 alpha (PI4KIIalpha). We previously reported that this membrane-anchored enzyme is a regulator of AP-3 recruitment to membranes and a cargo of AP-3 ( Craige, B., Salazar, G., and Faundez, V. (2008) Mol. Biol. Cell 19, 1415-1426 ). Using cells deficient in different Hermansky-Pudlak syndrome complexes, we identified that BLOC-1, but not BLOC-2 or BLOC-3, deficiencies affect PI4KIIalpha inclusion into AP-3 complexes. BLOC-1, PI4KIIalpha, and AP-3 belong to a tripartite complex, and down-regulation of either PI4KIIalpha, BLOC-1, or AP-3 complexes led to similar LAMP1 phenotypes. Our analysis indicates that BLOC-1 complex modulates the association of PI4KIIalpha with AP-3. These results suggest that AP-3 and BLOC-1 act, either in concert or sequentially, to specify sorting of PI4KIIalpha along the endocytic route.
Highlights
(AP-1 to AP-4) are critical to generate vesicles of specific composition from the different organelles constituting the exocytic and endocytic routes [2,3,4]
Hermansky-Pudlak syndrome (HPS) Gene Deficiencies Selectively Affect the Composition of absence of BLOC-2 and BLOC-3 did not affect PI4KII␣ or the AP-3-BLOC-1-PI4KII␣ Protein Complexes—PI4KII␣, clathrin levels present in AP-3 cross-linked complexes (Fig. 6C, BLOC-1, BLOC-2, and HOPS complex subunits are present in Hps3coa/coa, Hps4le/le cells, lanes 5 and 5Ј)
A targeted search for subunits from other HPS complexes identified BLOC-2 and HOPS in non-neuronal cells (Figs. 5 and 6). These results provide novel insight into the molecular organization of HPS protein complexes indicating that AP-3 forms complexes with clathrin and cargoes, such as PI4KII␣, and three other HPS complexes, BLOC-1, -2, and HOPS
Summary
Interactions organizing these complexes and how they might control membrane protein targeting. We took advantage of cell-permeant and reversible cross-linking of HPS complexes followed by their immunoaffinity purification to identify novel molecular interactions. Cross-linked AP-3 co-purified with BLOC-1, BLOC-2, HOPS, clathrin, and the membrane protein PI4KII␣. Using mutant cells deficient in either individual HPS complexes or a combination of them, we found that BLOC-1 facilitates the interaction of AP-3 and PI4KII␣. BLOC-1, PI4KII␣, and AP-3 form a tripartite complex as demonstrated by sequential co-immunoprecipitations as well as by similar LAMP1 distribution phenotypes induced by down-regulation of components of this tripartite complex. Our findings indicate that BLOC-1 complex modulates the recognition of PI4KII␣ by AP-3. These data suggest that AP-3, either in concert or sequentially with BLOC-1, participates in the sorting of common membrane proteins along the endocytic route
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