Mutational analyses have revealed many genes that are required for proper biogenesis of lysosomes and lysosome-related organelles. The proteins encoded by these genes assemble into five distinct complexes (AP-3, BLOC-1-3, and HOPS) that either sort membrane proteins or interact with SNAREs. Several of these seemingly distinct complexes cause similar phenotypic defects when they are rendered defective by mutation, but the underlying cellular mechanism is not understood. Here, we show that the BLOC-1 complex resides on microvesicles that also contain AP-3 subunits and membrane proteins that are known AP-3 cargoes. Mouse mutants that cause BLOC-1 or AP-3 deficiencies affected the targeting of LAMP1, phosphatidylinositol-4-kinase type II alpha, and VAMP7-TI. VAMP7-TI is an R-SNARE involved in vesicle fusion with late endosomes/lysosomes, and its cellular levels were selectively decreased in cells that were either AP-3-or BLOC-1-deficient. Furthermore, BLOC-1 deficiency selectively altered the subcellular distribution of VAMP7-TI cognate SNAREs. These results indicate that the BLOC-1 and AP-3 protein complexes affect the targeting of SNARE and non-SNARE AP-3 cargoes and suggest a function of the BLOC-1 complex in membrane protein sorting. INTRODUCTIONMembrane enclosed organelles possess distinctive protein compositions maintained by vesicle formation and vesicle fusion mechanisms. Vesicles are formed by coat and coat accessory molecules that selectively regulate the concentration of specific membrane proteins into departing vesicles. Once formed, vesicle contents are delivered to target membranes by vesicle fusion. This process depends on the pairing of fusogenic membrane proteins generically known as R-or vesicle (v)-SNAREs and Q-or target (t)-SNAREs (Springer et al., 1999;Bonifacino and Glick, 2004;Hong, 2005). Vesicle formation by coats and fusion by SNAREs is likely to be coordinately regulated, as suggested by the specific subcellular localizations that coats and SNAREs possess (Robinson, 2004;Hong, 2005). Predictably, coats either interact with and/or sort SNAREs. However, SNARE sorting mechanisms have been described only for a limited number of the SNAREs known to eukaryotic cells (Gurkan et al., 2005). For example, COPII interacts/sorts the R-(v)-SNAREs Bos1p and Bet1p into vesicles (Matsuoka et al., 1998;Springer and Schekman, 1998); the adaptors GGA1-2 sort the yeast Q-(t)-SNARE Pep12p (Black and Pelham, 2000); epsinR sorts/interacts with the Q-(t)-SNARE Vti1b (Hirst et al., 2004); the adaptor complex AP-3 sorts or interacts with the R-(v)-SNAREs VAMP7-TI (Martinez-Arca et al., 2003) and engineered variants of VAMP2 (Salem et al., 1998); and the adaptor complex AP-1 sorts/interacts with VAMP4 (Peden et al., 2001). These coat-SNARE interactions define prebudding interactions of the vesicle sorting and fusion machineries. However, it is poorly understood in vertebrates whether 1) these interactions persist at later stages once a vesicle is formed and cargo concentration is completed and 2) if molecules or com...

Cytoskeletal networks control organelle subcellular distribution and function. Herein, we describe a previously unsuspected association between intermediate filament proteins and the adaptor complex AP-3. AP-3 and intermediate filament proteins cosedimented and coimmunoprecipitated as a complex free of microtubule and actin binding proteins. Genetic perturbation of the intermediate filament cytoskeleton triggered changes in the subcellular distribution of the adaptor AP-3 and late endocytic/lysosome compartments. Concomitant with these architectural changes, and similarly to AP-3-null mocha cells, fibroblasts lacking vimentin were compromised in their vesicular zinc uptake, their organellar pH, and their total and surface content of AP-3 cargoes. However, the total content and surface levels, as well as the distribution of the transferrin receptor, a membrane protein whose sorting is AP-3 independent, remained unaltered in both AP-3- and vimentin-null cells. Based on the phenotypic convergence between AP-3 and vimentin deficiencies, we predicted and documented a reduced autophagosome content in mocha cells, a phenotype previously reported in cells with disrupted intermediate filament cytoskeletons. Our results reveal a novel role of the intermediate filament cytoskeleton in organelle/adaptor positioning and in regulation of the adaptor complex AP-3.

Adaptor protein (AP)-2 and AP-3-dependent mechanisms control the sorting of membrane proteins into synaptic vesicles. Mouse models deficient in AP-3, mocha, develop a neurological phenotype of which the central feature is an alteration of the luminal synaptic vesicle composition. This is caused by a severe reduction of vesicular levels of the zinc transporter 3 (ZnT3). It is presently unknown whether this mocha defect is restricted to ZnT3 or encompasses other synaptic vesicle proteins capable of modifying synaptic vesicle contents, such as transporters or channels. In this study, we identified a chloride channel, ClC-3, whose level in synaptic vesicles and hippocampal mossy fiber terminals was reduced in the context of the mocha AP-3 deficiency. In PC-12 cells, ClC-3 was present in transferrin receptorpositive endosomes, where it was targeted to synapticlike microvesicles (SLMV) by a mechanism sensitive to brefeldin A, a signature of the AP-3-dependent route of SLMV biogenesis. ClC-3 was packed in SLMV along with the AP-3-targeted synaptic vesicle protein ZnT3. Co-segregation of ClC-3 and ZnT3 to common intracellular compartments was functionally significant as revealed by increased vesicular zinc transport with increased ClC3 expression. Our work has identified a synaptic vesicle protein in which trafficking to synaptic vesicles is regulated by AP-3. In addition, our findings indicate that ClC-3 and ZnT3 reside in a common vesicle population where they functionally interact to determine vesicle luminal composition.

Actin, one of the major filamentous cytoskeletal molecules, is involved in a variety of cellular functions. Whereas an association between muscle actin mutations and skeletal and cardiac myopathies has been well documented, reports of human disease arising from mutations of nonmuscle actin genes have been rare. We have identified a missense point mutation in the gene coding for beta -actin that results in an arginine-to-tryptophan substitution at position 183. The disease phenotype includes developmental midline malformations, sensory hearing loss, and a delayed-onset generalized dystonia syndrome in monozygotic twins. Cellular studies of a lymphoblastoid cell line obtained from an affected patient demonstrated morphological abnormalities of the actin cytoskeleton and altered actin depolymerization dynamics in response to latrunculin A, an actin monomer-sequestering drug. Resistance to latrunculin A was also observed in NIH 3T3 cells expressing the mutant actin. These findings suggest that mutations in nonmuscle actins may be associated with a broad spectrum of developmental malformations and/or neurological abnormalities such as dystonia.

During the last two decades, much attention has been focused on the regulation of membrane traffic by the actin and microtubule cytoskeletal networks. Their dynamic and polarized behavior and associated motors provide a logical framework from which architectural and movement cues can be communicated to organelles. The study of these cytoskeletal systems has been greatly aided by pharmacological agents. In contrast, intermediate filaments (IFs) have largely been neglected as a potential player in membrane traffic, both because a comprehensive pharmacology to perturb them does not exist and because they lack the intrinsic polarity and specific motors that make the other cytoskeletal systems attractive. In this review, we will discuss evidence suggesting that IFs may play roles in controlling organelle positioning and in membrane protein targeting. Furthermore, we will discuss potential mechanisms by which IFs may regulate the localization and function of organelles.