Article

Hermansky-Pudlak syndrome protein complexes associate with phosphatidylinositol 4-kinase type II alpha in neuronal and non-neuronal cells.

Department of Cell Biology and Medicine, Emory University, Atlanta, Georgia 30322, USA.
Journal of Biological Chemistry (Impact Factor: 4.6). 12/2008; 284(3):1790-802. DOI: 10.1074/jbc.M805991200
Source: PubMed

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.

0 Bookmarks
 · 
93 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: Specificity of membrane fusion in vesicular trafficking is dependent on proper subcellular distribution of soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs). Although SNARE complexes are fairly promiscuous in vitro, significant specificity is achieved in cells due to spatial segregation and shielding of SNARE motifs prior to association with cognate Q-SNAREs. In this study we identified phosphatidylinositol 4-kinase IIα (PI4K2A) as a binding partner of vesicle-associated membrane protein 3 (VAMP3), a small R-SNARE involved in recycling and retrograde transport, and found that the two proteins co-reside on tubulo-vesicular endosomes. PI4K2A knockdown inhibited VAMP3 trafficking to perinuclear membranes and impaired the rate of VAMP3-mediated recycling of the transferrin receptor. Moreover, depletion of PI4K2A significantly decreased association of VAMP3 with its cognate Q-SNARE, Vti1a. Although binding of VAMP3 to PI4K2A did not require kinase activity, acute depletion of PtdIns4P on endosomes significantly delayed VAMP3 trafficking. Phospholipid modulation of SNARE function has been proposed based on in vitro studies and our study provides mechanistic evidence in support of these claims by identifying PI4K2A and PtdIns4P as regulators of an R-SNARE in intact cells.
    Journal of cell science. 07/2014;
  • [Show abstract] [Hide abstract]
    ABSTRACT: The gene DTNBP1 encodes the protein dysbindin and is among the most promising and highly investigated schizophrenia-risk genes. Accumulating evidence suggests that dysbindin plays an important role in the regulation of neuroplasticity. Dysbindin was reported to be a stable component of BLOC-1 complex in cytosol. However, little is known about the endogenous dysbindin-containing complex in the synaptosome of brain. In this study, we investigated the associated proteome of dysbindin in the P2 synaptosome fraction of mouse brain. Our data suggest that dysbindin has three isoforms associating with different complexes in the P2 of mouse brain. To facilitate immuno-purification, BAC transgenic mice expressing a tagged dysbindin were generated, and 47 putative dysbindin-associated proteins, including all components in BLOC-1, were identified by mass spectrometry in the dysbindin-containing complex purified from P2. The interactions of several selected candidates, including WDR11, FAM91A1, snapin, muted, pallidin and two proteasome subunits PSMD9 and PSMA4, were verified by co-immunoprecipitation. The specific proteasomal activity is significantly reduced in the P2 fraction of the brains of the dysbindin null mutant (sandy) mice. Our data suggest that dysbindin is functionally interrelated to the ubiquitin-proteasome system and offer a molecular repertoire for future study of dysbindin functional networks in brain.
    Journal of Proteome Research 09/2014; · 5.06 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Exocytotic release of glutamate depends upon loading of the neurotransmitter into synaptic vesicles by vesicular glutamate transporters, VGLUTs. The major isoforms, VGLUT1 and 2, exhibit a complementary pattern of expression in synapses of the adult rodent brain that correlates with the probability of release and potential for plasticity. Indeed, expression of different VGLUT protein isoforms confers different properties of release probability. Expression of VGLUT1 or 2 protein also determines the kinetics of synaptic vesicle recycling. To identify molecular determinants that may be related to reported differences in VGLUT trafficking and glutamate release properties, we investigated some of the intrinsic differences between the two isoforms. VGLUT1 and 2 exhibit a high degree of sequence homology, but differ in their N- and C-termini. While the C-termini of VGLUT1 and 2 share a dileucine-like trafficking motif and a proline-, glutamate-, serine-, and threonine-rich PEST domain, only VGLUT1 contains two polyproline domains and a phosphorylation consensus sequence in a region of acidic amino acids. The interaction of a VGLUT1 polyproline domain with the endocytic protein endophilin recruits VGLUT1 to a fast recycling pathway. To identify trans-acting cellular proteins that interact with the distinct motifs found in the C-terminus of VGLUT1, we performed a series of in vitro biochemical screening assays using the region encompassing the polyproline motifs, phosphorylation consensus sites, and PEST domain. We identify interactors that belong to several classes of proteins that modulate cellular function, including actin cytoskeletal adaptors, ubiquitin ligases, and tyrosine kinases. The nature of these interactions suggests novel avenues to investigate the modulation of synaptic vesicle protein recycling.
    PLoS ONE 10/2014; 9(10):e109824. · 3.53 Impact Factor

Full-text

Download
35 Downloads
Available from
May 26, 2014