Parkinson’s disease (PD) is characterized pathologically by intraneuronal inclusions called Lewy bodies, largely comprised of α-synuclein. Multiplication the α-synuclein gene locus increases expression and causes PD. Thus, overexpression wild-type toxic. In this study, we demonstrate that impairs macroautophagy in mammalian cells transgenic mice. Our data show compromises autophagy via Rab1a inhibition rescues defect caused Inhibition or knockdown mislocalization protein, Atg9, decreases...

The adaptor protein (AP) 3 complex has been implicated in the transport of lysosomal membrane proteins, but its precise site action remained controversial. Here, we show by immuno-electron microscopy that AP-3 is associated with budding profiles evolving from a tubular endosomal compartment also exhibits positive for AP-1. colocalizes clathrin, to lesser extent than does AP-3- and AP-1-bearing compartments contain endocytosed transferrin, transferrin receptor, asialoglycoprotein low amounts...

We have recently shown that two proteins related to of the adaptor subunits clathrincoated vesicles, p47 (μ3) and β-NAP (β3B), are part an adaptor-like complex not associated with clathrin (Simpson, F., N.A. Bright, M.A. West, L.S. Newman, R.B. Darnell, M.S. Robinson, 1996. J. Cell Biol. 133:749–760). In present study we searched EST database identified, cloned, sequenced a ubiquitously expressed homologue β-NAP, β3A, as well homologues α/γ σ subunits, δ σ3, which also expressed. Antibodies...

An integral part of cell division is the separation daughter cells via cytokinesis. There now good evidence that completion cytokinesis requires coordinated membrane trafficking to deliver new tip furrow and complete abscission. Here we have examined traffic in describe several novel observations. First, show Rab11- FIP3-containing recycling endosomes accumulate near cleavage are required for successful Second, demonstrate Rab11-FIP3 protein complex intimately involved delivery furrow....

Specialized cells exploit adaptor protein complexes for unique post-Golgi sorting events, providing a model system to specify function. Here, we show that AP-3 and AP-1 function independently in of the melanocyte-specific tyrosinase from endosomes melanosome, specialized lysosome-related organelle distinguishable lysosomes. localize melanocytes primarily clathrin-coated buds on tubular early near melanosomes. Both adaptors recognize dileucine-based melanosome signal, largely colocalizes with...

Rab GTPases are localized to various intracellular compartments and known play important regulatory roles in membrane trafficking. Here, we report the subcellular distribution function of Rab14. By immunofluorescence immunoelectron microscopy, both endogenous as well overexpressed Rab14 were biosynthetic (rough endoplasmic reticulum, Golgi, trans-Golgi network) endosomal (early vacuoles associated vesicles). Notably overexpression Rab14Q70L shifted toward early endosome vesicles, whereas...

SNAREs provide a large part of the specificity and energy needed for membrane fusion and, to do so, must be localized their correct membranes. Here, we show that R-SNAREs VAMP8, VAMP3, VAMP2, which cycle between plasma endosomes, bind directly ubiquitously expressed, PtdIns4,5P2-binding, endocytic clathrin adaptor CALM/PICALM. X-ray crystallography shows N-terminal halves SNARE motifs CALMANTH domain as helices in manner mimics complex formation. Mutation residues CALM:SNARE interface...

Although adaptor protein complex 1 (AP-1) and Golgi-localized, γ ear-containing, ADP-ribosylation factor-binding proteins (GGAs) are both adaptors for clathrin-mediated intracellular trafficking, the pathways they mediate their relationship to each other remain open questions [1Bonifacino J.S. The GGA proteins: on move.Nat. Rev. Mol. Cell Biol. 2004; 5: 23-32Crossref PubMed Scopus (303) Google Scholar]. To tease apart functions of AP-1 GGAs, we rapidly inactivated using "knocksideways"...

Invadopodia-dependent degradation of the basement membrane plays a major role during metastasis breast cancer cells. Basement is mediated by targeted secretion various matrix metalloproteinases (MMPs). Specifically, MMP2 and MMP9 (MMP2/9) possess ability to hydrolyze components regulate aspects tumor growth metastasis. However, transport machinery that mediates targeting MMP2/9 invadopodia cell invasion remains be defined. Because Rab GTPases are key regulators transport, we screened human...

The role of SNAREs in mammalian constitutive secretion remains poorly defined. To address this, we have developed a novel flow cytometry-based assay for measuring and performed targeted SNARE Sec1/Munc18 (SM) protein-specific siRNA screen (38 SNAREs, 4 SNARE-like proteins 7 SM proteins). We identified the endoplasmic reticulum (ER)/Golgi syntaxin 5, 17, 18, GS27, SLT1, Sec20, Sec22b, Ykt6 protein Sly1, along with post-Golgi SNAP-29 19, as being required secretion. Depletion or 19 causes...

Macrophages are critical effectors of the early innate response to bacteria in tissues. Phagocytosis and killing interrelated functions essential for bacterial clearance but rate-limiting step when macrophages challenged with large numbers major medical pathogen Staphylococcus aureus is unknown. We show that have a finite capacity intracellular fail match sustained phagocytosis microbial exposed inocula S. (Newman, SH1000 USA300 strains). ingestion by associated rapid decline viability...

The mouse mutants mocha and pearl are deficient in the AP-3 δ β3A subunits, respectively. We have used cells from these mice to investigate both assembly of complexes function. In cells, β3 μ3 subunits coassemble into a heterodimer, whereas σ3 subunit remains monomeric. gets destroyed. yeast two hybrid system was confirm interactions, also demonstrate that A (ubiquitous) B (neuronal-specific) isoforms can interact with each other. Pearl cell lines were generated express β3A, β3B, β3Aβ2...

Cytoskeletal networks control organelle subcellular distribution and function. Herein, we describe a previously unsuspected association between intermediate filament proteins the adaptor complex AP-3. AP-3 cosedimented coimmunoprecipitated as free of microtubule actin binding proteins. Genetic perturbation cytoskeleton triggered changes in late endocytic/lysosome compartments. Concomitant with these architectural changes, similarly to AP-3-null mocha cells, fibroblasts lacking vimentin were...

Mutational analyses have revealed many genes that are required for proper biogenesis of lysosomes and lysosome-related organelles. The proteins encoded by these assemble into five distinct complexes (AP-3, BLOC-1-3, HOPS) either sort membrane or interact with SNAREs. Several seemingly cause similar phenotypic defects when they rendered defective mutation, but the underlying cellular mechanism is not understood. Here, we show BLOC-1 complex resides on microvesicles also contain AP-3 subunits...

Mammalian cells acquire cholesterol mainly from LDL. LDL enter the endosomes, allowing cholesteryl esters to be hydrolyzed by acid lipase. The (LDL-CHOL) enters Niemann–Pick type C1 (NPC1)-containing endosomal compartment en route various destinations. Whether Golgi is involved in LDL-CHOL transport downstream of NPC1 has not been demonstrated. Using subcellular fractionation and immunoadsorption enrich for specific membrane fractions, here we show that, when parental Chinese hamster ovary...

Endocytosis is crucial for various aspects of cell homeostasis. Here, we show that proapoptotic death receptors (DRs) trigger selective destruction the clathrin-dependent endocytosis machinery. DR stimulation induced rapid, caspase-mediated cleavage key clathrin-pathway components, halting cellular uptake classic cargo protein transferrin. DR-proximal initiator caspases cleaved clathrin adaptor subunit AP2alpha between functionally distinct domains, whereas effector processed clathrin's...

Autophagy is a lysosome-dependent cellular catabolic mechanism that mediates the turnover of intracellular organelles and long-lived proteins. Reduced autophagic activity has been shown to lead accumulation misfolded proteins in neurons might be involved chronic neurodegenerative diseases. Here, we uncover an essential role for syntaxin-5 SNARE complex autophagy. Using genetic knockdown, show regulates later stages autophagy after initial formation autophagosomes. This acts on by regulating...

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 which central feature is an alteration luminal vesicle composition. This caused by severe reduction vesicular levels zinc transporter 3 (ZnT3). It presently unknown whether this mocha defect restricted to ZnT3 or encompasses other capable modifying contents, such as transporters channels. In study, we...

The Rab11 subfamily of GTPases plays an important role in vesicle trafficking from endosomes to the plasma membrane. At least six effectors (family Rab11-interacting proteins (FIPs)) have been shown interact with and are hypothesized regulate various membrane pathways such as transferrin recycling, cytokinesis, epidermal growth factor trafficking. In this study, we characterized interactions FIPs GTPase using isothermal titration calorimetric studies mutational analysis. Our data suggest...

Rab 11 GTPase is an important regulator of endocytic membrane traffic. Recently, we and others have identified a novel family Rab11 binding proteins, known as Rab11-family interacting proteins (FIPs). One the members, coupling protein (RCP), was to both Rab4 GTPases. RCP therefore suggested serve dual function protein. In this study, characterized cellular functions mapped its interactions with Rab11. Our data show that interacts only weakly in vitro does not play role vivo. Furthermore, our...