scholarly journals BLOC-1 Complex Deficiency Alters the Targeting of Adaptor Protein Complex-3 Cargoes

2006 ◽  
Vol 17 (9) ◽  
pp. 4014-4026 ◽  
Author(s):  
G. Salazar ◽  
B. Craige ◽  
M. L. Styers ◽  
K. A. Newell-Litwa ◽  
M. M. Doucette ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Protein Complexes ◽  
Adaptor Protein ◽  
Cellular Mechanism ◽  
Type Ii ◽  
Mouse Mutants ◽  
Late Endosomes ◽  
Complex Deficiency ◽  
Lysosome Related Organelles ◽  
Phosphatidylinositol 4

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.

scholarly journals Phosphatidylinositol-4-Kinase Type II Alpha Contains an AP-3–sorting Motif and a Kinase Domain That Are Both Required for Endosome Traffic

2008 ◽  
Vol 19 (4) ◽  
pp. 1415-1426 ◽  
Author(s):  
Branch Craige ◽  
Gloria Salazar ◽  
Victor Faundez
Keyword(s):  
Synaptic Vesicles ◽  
Kinase Activity ◽  
Kinase Domain ◽  
Membrane Domains ◽  
Type Ii ◽  
Catalytic Domains ◽  
Sorting Motif ◽  
Lysosome Related Organelles ◽  
Mutant Phenotypes ◽  
Phosphatidylinositol 4

The adaptor complex 3 (AP-3) targets membrane proteins from endosomes to lysosomes, lysosome-related organelles and synaptic vesicles. Phosphatidylinositol-4-kinase type II α (PI4KIIα) is one of several proteins possessing catalytic domains that regulate AP-3–dependent sorting. Here we present evidence that PI4KIIα uniquely behaves both as a membrane protein cargo as well as an enzymatic regulator of adaptor function. In fact, AP-3 and PI4KIIα form a complex that requires a dileucine-sorting motif present in PI4KIIα. Mutagenesis of either the PI4KIIα-sorting motif or its kinase-active site indicates that both are necessary to interact with AP-3 and properly localize PI4KIIα to LAMP-1–positive endosomes. Similarly, both the kinase activity and the sorting signal present in PI4KIIα are necessary to rescue endosomal PI4KIIα siRNA-induced mutant phenotypes. We propose a mechanism whereby adaptors use canonical sorting motifs to selectively recruit a regulatory enzymatic activity to restricted membrane domains.

scholarly journals The WASH complex, an endosomal Arp2/3 activator, interacts with the Hermansky–Pudlak syndrome complex BLOC-1 and its cargo phosphatidylinositol-4-kinase type IIα

2013 ◽  
Vol 24 (14) ◽  
pp. 2269-2284 ◽  
Author(s):  
P. V. Ryder ◽  
R. Vistein ◽  
A. Gokhale ◽  
M. N. Seaman ◽  
M. A. Puthenveedu ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Adaptor Protein ◽  
Coat Proteins ◽  
Lipid Kinase ◽  
Rho Family Gtpases ◽  
Vesicle Biogenesis ◽  
Hermansky Pudlak Syndrome ◽  
Rho Family ◽  
Lysosome Related Organelles ◽  
Phosphatidylinositol 4

Vesicle biogenesis machinery components such as coat proteins can interact with the actin cytoskeleton for cargo sorting into multiple pathways. It is unknown, however, whether these interactions are a general requirement for the diverse endosome traffic routes. In this study, we identify actin cytoskeleton regulators as previously unrecognized interactors of complexes associated with the Hermansky–Pudlak syndrome. Two complexes mutated in the Hermansky–Pudlak syndrome, adaptor protein complex-3 and biogenesis of lysosome-related organelles complex-1 (BLOC-1), interact with and are regulated by the lipid kinase phosphatidylinositol-4-kinase type IIα (PI4KIIα). We therefore hypothesized that PI4KIIα interacts with novel regulators of these complexes. To test this hypothesis, we immunoaffinity purified PI4KIIα from isotope-labeled cell lysates to quantitatively identify interactors. Strikingly, PI4KIIα isolation preferentially coenriched proteins that regulate the actin cytoskeleton, including guanine exchange factors for Rho family GTPases such as RhoGEF1 and several subunits of the WASH complex. We biochemically confirmed several of these PI4KIIα interactions. Of importance, BLOC-1 complex, WASH complex, RhoGEF1, or PI4KIIα depletions altered the content and/or subcellular distribution of the BLOC-1–sensitive cargoes PI4KIIα, ATP7A, and VAMP7. We conclude that the Hermansky–Pudlak syndrome complex BLOC-1 and its cargo PI4KIIα interact with regulators of the actin cytoskeleton.

scholarly journals Phosphatidylinositol-4-Kinase Type II α Is a Component of Adaptor Protein-3-derived Vesicles

2005 ◽  
Vol 16 (8) ◽  
pp. 3692-3704 ◽  
Author(s):  
Gloria Salazar ◽  
Branch Craige ◽  
Bruce H. Wainer ◽  
Jun Guo ◽  
Pietro De Camilli ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Pc12 Cells ◽  
Membrane Fraction ◽  
Functional Relationship ◽  
Adaptor Protein ◽  
Mossy Fibers ◽  
Nerve Terminals ◽  
Type Ii ◽  
Phosphatidylinositol 4 ◽  
Lysosomal Proteins

A membrane fraction enriched in vesicles containing the adaptor protein (AP) -3 cargo zinc transporter 3 was generated from PC12 cells and was used to identify new components of these organelles by mass spectrometry. Proteins prominently represented in the fraction included AP-3 subunits, synaptic vesicle proteins, and lysosomal proteins known to be sorted in an AP-3-dependent way or to interact genetically with AP-3. A protein enriched in this fraction was phosphatidylinositol-4-kinase type IIα (PI4KIIα). Biochemical, pharmacological, and morphological analyses supported the presence of PI4KIIα in AP-3-positive organelles. Furthermore, the subcellular localization of PI4KIIα was altered in cells from AP-3-deficient mocha mutant mice. The PI4KIIα normally present both in perinuclear and peripheral organelles was substantially decreased in the peripheral membranes of AP-3-deficient mocha fibroblasts. In addition, as is the case for other proteins sorted in an AP-3-dependent way, PI4KIIα content was strongly reduced in nerve terminals of mocha hippocampal mossy fibers. The functional relationship between AP-3 and PI4KIIα was further explored by PI4KIIα knockdown experiments. Reduction of the cellular content of PI4KIIα strongly decreased the punctate distribution of AP-3 observed in PC12 cells. These results indicate that PI4KIIα is present on AP-3 organelles where it regulates AP-3 function.

scholarly journals Nanobody-dependent delocalization of endocytic machinery in Arabidopsis root cells dampens their internalization capacity

2020 ◽  
Author(s):  
Joanna Winkler ◽  
Andreas De Meyer ◽  
Evelien Mylle ◽  
Peter Grones ◽  
Veronique Storme ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Protein Complexes ◽  
Function Analysis ◽  
Plant Cells ◽  
Adaptor Protein ◽  
Genetic Mutation ◽  
Loss Of Function ◽  
Expression Domain ◽  
Specific Expression ◽  
Root Cells

AbstractPlant cells perceive and adapt to an ever-changing environment by modifying their plasma membrane (PM) proteome. Whereas secretion deposits new integral membrane proteins, internalization by endocytosis removes membrane proteins and associated ligands, largely with the aid of adaptor protein complexes and the scaffolding molecule clathrin. Two adaptor protein complexes function in clathrin-mediated endocytosis at the PM in plant cells, the heterotetrameric Adaptor Protein 2 (AP-2) complex and the octameric TPLATE complex (TPC). Whereas single subunit mutants in AP-2 develop into viable plants, genetic mutation of a single TPC subunit causes fully penetrant male sterility and silencing single subunits leads to seedling lethality. To address TPC function in somatic root cells, while minimizing indirect effects on plant growth, we employed nanobody-dependent delocalization of a functional, GFP-tagged TPC subunit, TML, in its respective homozygous genetic mutant background. In order to decrease the amount of functional TPC at the PM, we targeted our nanobody construct to the mitochondria and fused it to TagBFP2 to visualize it independently of its bait. We furthermore limited the effect of our delocalization to those tissues that are easily accessible for live-cell imaging by expressing it from the PIN2 promotor, which is active in root epidermal and cortex cells. With this approach, we successfully delocalized TML from the PM. Moreover, we also show co-recruitment of TML-GFP and AP2A1-TagRFP to the mitochondria, suggesting that our approach delocalized complexes, rather than individual adaptor complex subunits. In line with the specific expression domain, we only observed minor effects on root growth, yet realized a clear reduction of endocytic flux in epidermal root cells. Nanobody-dependent delocalization in plants, here exemplified using a TPC subunit, has the potential to be widely applicable to achieve specific loss-of-function analysis of otherwise lethal mutants.

scholarly journals An early endosome–derived retrograde trafficking pathway promotes secretory granule maturation

2020 ◽  
Vol 219 (3) ◽  
Author(s):  
Cheng-I J. Ma ◽  
Yitong Yang ◽  
Taeah Kim ◽  
Chang Hua Chen ◽  
Gordon Polevoy ◽  
...  
Keyword(s):  
Secretory Granules ◽  
Biologically Active ◽  
Type Ii ◽  
Endosomal Sorting ◽  
Trafficking Pathway ◽  
Granule Maturation ◽  
Lysosome Related Organelles ◽  
Phosphatidylinositol 4 ◽  
Endosomal System ◽  
External Stimuli

Regulated secretion is a fundamental cellular process in which biologically active molecules stored in long-lasting secretory granules (SGs) are secreted in response to external stimuli. Many studies have described mechanisms responsible for biogenesis and secretion of SGs, but how SGs mature remains poorly understood. In a genetic screen, we discovered a large number of endolysosomal trafficking genes required for proper SG maturation, indicating that maturation of SGs might occur in a manner similar to lysosome-related organelles (LROs). CD63, a tetraspanin known to decorate LROs, also decorates SG membranes and facilitates SG maturation. Moreover, CD63-mediated SG maturation requires type II phosphatidylinositol 4 kinase (PI4KII)-dependent early endosomal sorting and accumulation of phosphatidylinositol 4-phosphate (PI4P) on SG membranes. In addition, the PI4P effector Past1 is needed for formation of stable PI4KII-containing endosomal tubules associated with this process. Our results reveal that maturation of post-Golgi–derived SGs requires trafficking via the endosomal system, similar to mechanisms employed by LROs.

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

2008 ◽  
Vol 284 (3) ◽  
pp. 1790-1802 ◽  
Author(s):  
Gloria Salazar ◽  
Stephanie Zlatic ◽  
Branch Craige ◽  
Andrew A. Peden ◽  
Jan Pohl ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Neuronal Cells ◽  
Type Ii ◽  
Hermansky Pudlak Syndrome ◽  
Phosphatidylinositol 4 ◽  
Syndrome Protein

scholarly journals Roles of BLOC-1 and Adaptor Protein-3 Complexes in Cargo Sorting to Synaptic Vesicles

2009 ◽  
Vol 20 (5) ◽  
pp. 1441-1453 ◽  
Author(s):  
Karen Newell-Litwa ◽  
Gloria Salazar ◽  
Yoland Smith ◽  
Victor Faundez
Keyword(s):  
Membrane Proteins ◽  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Adaptor Protein ◽  
Protein Composition ◽  
Organelle Biogenesis ◽  
Vesicle Membrane ◽  
Early Endosomes ◽  
Lysosomal Sorting ◽  
Lysosome Related Organelles

Neuronal lysosomes and their biogenesis mechanisms are primarily thought to clear metabolites and proteins whose abnormal accumulation leads to neurodegenerative disease pathology. However, it remains unknown whether lysosomal sorting mechanisms regulate the levels of membrane proteins within synaptic vesicles. Using high-resolution deconvolution microscopy, we identified early endosomal compartments where both selected synaptic vesicle and lysosomal membrane proteins coexist with the adaptor protein complex 3 (AP-3) in neuronal cells. From these early endosomes, both synaptic vesicle membrane proteins and characteristic AP-3 lysosomal cargoes can be similarly sorted to brain synaptic vesicles and PC12 synaptic-like microvesicles. Mouse knockouts for two Hermansky–Pudlak complexes involved in lysosomal biogenesis from early endosomes, the ubiquitous isoform of AP-3 (Ap3b1−/−) and muted, defective in the biogenesis of lysosome-related organelles complex 1 (BLOC-1), increased the content of characteristic synaptic vesicle proteins and known AP-3 lysosomal proteins in isolated synaptic vesicle fractions. These phenotypes contrast with those of the mouse knockout for the neuronal AP-3 isoform involved in synaptic vesicle biogenesis (Ap3b2−/−), in which the content of select proteins was reduced in synaptic vesicles. Our results demonstrate that lysosomal and lysosome-related organelle biogenesis mechanisms regulate steady-state synaptic vesicle protein composition from shared early endosomes.

scholarly journals PI4P and BLOC-1 remodel endosomal membranes into tubules

2021 ◽  
Author(s):  
Riddhi Atul Jani ◽  
Aurelie Di Cicco ◽  
Tal Keren-Kaplan ◽  
Silvia Vale-Costa ◽  
Daniel Hamaoui ◽  
...  
Keyword(s):  
Intracellular Trafficking ◽  
Membrane Lipids ◽  
Intracellular Pathogens ◽  
Type Ii ◽  
Membrane Remodeling ◽  
Membrane Systems ◽  
Endosomal Membranes ◽  
Lysosome Related Organelles ◽  
Phosphatidylinositol 4

Intracellular trafficking is mediated by transport carriers that originate by membrane remodeling from donor organelles. Tubular carriers play major roles in the flux of membrane lipids and proteins to acceptor organelles. However, how lipids and proteins impose a tubular geometry on the carriers is incompletely understood. By exploiting imaging approaches at different scales on cells and in vitro membrane systems, we show that phosphatidylinositol-4-phosphate (PI4P) and biogenesis of lysosome-related organelles complex 1 (BLOC-1) govern the formation, stability and functions of recycling endosomal tubules. Endosomal PI4P production by type II PI4-kinases is needed to form nascent curved tubules through binding of BLOC-1 that stabilize and elongate them. Membrane remodeling by the PI4P/ BLOC-1 module functions not only in the recycling of endosomal cargoes, but also in the lifecycles of intracellular pathogens such as Chlamydia bacteria and influenza virus. This study demonstrates how a phospholipid and a protein complex coordinate as a minimal machinery to remodel cellular membranes into functional tubes.

scholarly journals Role of AP1 and Gadkin in the traffic of secretory endo-lysosomes

2011 ◽  
Vol 22 (12) ◽  
pp. 2068-2082 ◽  
Author(s):  
Karine Laulagnier ◽  
Nicole L. Schieber ◽  
Tanja Maritzen ◽  
Volker Haucke ◽  
Robert G. Parton ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Secretory Pathway ◽  
Protein Complexes ◽  
Adaptor Protein ◽  
Cytosolic Calcium ◽  
Endocytic Pathway ◽  
Secretory Process ◽  
Microtubule Depolymerization ◽  
Lysosome Related Organelles ◽  
Endocytic Compartments

Whereas lysosome-related organelles (LRO) of specialized cells display both exocytic and endocytic features, lysosomes in nonspecialized cells can also acquire the property to fuse with the plasma membrane upon an acute rise in cytosolic calcium. Here, we characterize this unconventional secretory pathway in fibroblast-like cells, by monitoring the appearance of Lamp1 on the plasma membrane and the release of lysosomal enzymes into the medium. After sequential ablation of endocytic compartments in living cells, we find that donor membranes primarily derive from a late compartment, but that an early compartment is also involved. Strikingly, this endo-secretory process is not affected by treatments that inhibit endosome dynamics (microtubule depolymerization, cholesterol accumulation, overexpression of Rab7 or its effector Rab-interacting lysosomal protein [RILP], overexpression of Rab5 mutants), but depends on Rab27a, a GTPase involved in LRO secretion, and is controlled by F-actin. Moreover, we find that this unconventional endo-secretory pathway requires the adaptor protein complexes AP1, Gadkin (which recruits AP1 by binding to the γ1 subunit), and AP2, but not AP3. We conclude that a specific fraction of the AP2-derived endocytic pathway is dedicated to secretory purposes under the control of AP1 and Gadkin.

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