Adaptor Protein CD2AP and L-type Lectin LMAN2 Regulate Exosome Cargo Protein Trafficking through the Golgi Complex

Glycosylphosphatidylinositol (GPI)-anchored proteins exit the ER in distinct vesicles from other secretory proteins, and this sorting event requires the Rab GTPase Ypt1p, tethering factors Uso1p, and the conserved oligomeric Golgi complex. Here we show that proper sorting depended on the vSNAREs, Bos1p, Bet1p, and Sec22p. However, the t-SNARE Sed5p was not required for protein sorting upon ER exit. Moreover, the sorting defect observed in vitro with bos1–1 extracts was also observed in vivo and was visualized by EM. Finally, transport and maturation of the GPI-anchored protein Gas1p was specifically affected in a bos1–1 mutant at semirestrictive temperature. Therefore, we propose that v-SNAREs are part of the cargo protein sorting machinery upon exit from the ER and that a correct sorting process is necessary for proper maturation of GPI-anchored proteins.

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Phosphoserine acidic cluster motifs in the cytoplasmic domains of transmembrane proteins bind distinct basic regions on the μ subunits of clathrin adaptor protein complexes

10.1101/286633 ◽

2018 ◽

Author(s):

Rajendra Singh ◽

Charlotte Stoneham ◽

Christopher Lim ◽

Xiaofei Jia ◽

Javier Guenaga ◽

...

Keyword(s):

Protein Trafficking ◽

Transmembrane Proteins ◽

Adaptor Protein ◽

Cellular Protein ◽

Membrane Lipid ◽

Dependent Manner ◽

Cytoplasmic Domains ◽

Clathrin Adaptor ◽

Hiv 1

AbstractProtein trafficking in the endosomal system involves the recognition of specific signals within the cytoplasmic domains (CDs) of transmembrane proteins by clathrin adaptors. One such signal is the phosphoserine acidic cluster (PSAC), the prototype of which is in the endoprotease Furin. How PSACs are recognized by clathrin adaptors has been controversial. We reported previously that HIV-1 Vpu, which modulates cellular immunoreceptors, contains a PSAC that binds to the µ subunits of clathrin adaptor protein (AP) complexes. Here, we show that the CD of Furin binds the µ subunits of AP-1 and AP-2 in a phosphorylation-dependent manner. Moreover, we identify a PSAC in a cytoplasmic loop of the cellular transmembrane Serinc3, an inhibitor of the infectivity of retroviruses. The two serines within the PSAC of Serinc3 are phosphorylated by casein kinase II and mediate interaction with the µ subunits in vitro. The sites of these serines vary among mammals in a manner consistent with host-pathogen conflict, yet the Serinc3-PSAC seems dispensible for anti-HIV activity and for counteraction by HIV-1 Nef. The CDs of Vpu, Furin, and the PSAC-containing loop of Serinc3 each bind the μ subunit of AP-2 (µ2) with similar affinities, but they appear to utilize different basic regions on µ2. The Serinc3 loop requires a region previously reported to bind the acidic plasma membrane lipid phosphatidylinositol-4,5-bisphosphate. These data suggest that the PSACs within different proteins recognize different basic regions on the µ surface, providing the potential to inhibit the activity of viral proteins without necessarily affecting cellular protein trafficking.

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The Abl/Enabled signaling pathway regulates Golgi architecture in Drosophila photoreceptor neurons

Molecular Biology of the Cell ◽

10.1091/mbc.e14-02-0729 ◽

2014 ◽

Vol 25 (19) ◽

pp. 2993-3005 ◽

Cited By ~ 8

Author(s):

Ramakrishnan Kannan ◽

Irina Kuzina ◽

Stephen Wincovitch ◽

Stephanie H. Nowotarski ◽

Edward Giniger

Keyword(s):

Cell Signaling ◽

Signaling Pathway ◽

Protein Trafficking ◽

Adaptor Protein ◽

Loss Of Function ◽

Abl Tyrosine Kinase ◽

Upstream Regulator ◽

Basal Portion ◽

Photoreceptor Neurons ◽

Drosophila Photoreceptor

The Golgi apparatus is optimized separately in different tissues for efficient protein trafficking, but we know little of how cell signaling shapes this organelle. We now find that the Abl tyrosine kinase signaling pathway controls the architecture of the Golgi complex in Drosophila photoreceptor (PR) neurons. The Abl effector, Enabled (Ena), selectively labels the cis-Golgi in developing PRs. Overexpression or loss of function of Ena increases the number of cis- and trans-Golgi cisternae per cell, and Ena overexpression also redistributes Golgi to the most basal portion of the cell soma. Loss of Abl or its upstream regulator, the adaptor protein Disabled, lead to the same alterations of Golgi as does overexpression of Ena. The increase in Golgi number in Abl mutants arises in part from increased frequency of Golgi fission events and a decrease in fusions, as revealed by live imaging. Finally, we demonstrate that the effects of Abl signaling on Golgi are mediated via regulation of the actin cytoskeleton. Together, these data reveal a direct link between cell signaling and Golgi architecture. Moreover, they raise the possibility that some of the effects of Abl signaling may arise, in part, from alterations of protein trafficking and secretion.

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Defective Pigment Granule Biogenesis and Aberrant Behavior Caused by Mutations in the Drosophila AP-3β Adaptin Gene ruby

Genetics ◽

10.1093/genetics/155.1.213 ◽

2000 ◽

Vol 155 (1) ◽

pp. 213-223

Author(s):

Doris Kretzschmar ◽

Burkhard Poeck ◽

Helmut Roth ◽

Roman Ernst ◽

Andreas Keller ◽

...

Keyword(s):

Protein Trafficking ◽

Coat Color ◽

Retinal Pigment ◽

Skin Pigmentation ◽

Pigment Granule ◽

Adaptor Protein ◽

Photoreceptor Cells ◽

Cell Types ◽

Eye Color ◽

Pigment Granules

Abstract Lysosomal protein trafficking is a fundamental process conserved from yeast to humans. This conservation extends to lysosome-like organelles such as mammalian melanosomes and insect eye pigment granules. Recently, eye and coat color mutations in mouse (mocha and pearl) and Drosophila (garnet and carmine) were shown to affect subunits of the heterotetrameric adaptor protein complex AP-3 involved in vesicle trafficking. Here we demonstrate that the Drosophila eye color mutant ruby is defective in the AP-3β subunit gene. ruby expression was found in retinal pigment and photoreceptor cells and in the developing central nervous system. ruby mutations lead to a decreased number and altered size of pigment granules in various cell types in and adjacent to the retina. Humans with lesions in the related AP-3βA gene suffer from Hermansky-Pudlak syndrome, which is caused by defects in a number of lysosome-related organelles. Hermansky-Pudlak patients have a reduced skin pigmentation and suffer from internal bleeding, pulmonary fibrosis, and visual system malfunction. The Drosophila AP-3β adaptin also appears to be involved in processes other than eye pigment granule biogenesis because all ruby allele combinations tested exhibited defective behavior in a visual fixation paradigm.

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A shared pathway of exosome biogenesis operates at plasma and endosome membranes

10.1101/545228 ◽

2019 ◽

Cited By ~ 5

Author(s):

Francis K. Fordjour ◽

George G. Daaboul ◽

Stephen J. Gould

Keyword(s):

Plasma Membrane ◽

Protein Trafficking ◽

Mechanistic Explanation ◽

Simple Explanation ◽

Common Pathway ◽

Exosome Biogenesis ◽

Cargo Protein ◽

Cargo Proteins ◽

Targeting Signal ◽

Membrane Bound

AbstractEukaryotic cells secrete exosomes, which are small (~30-200 nm dia.), single membrane-bound organelles that transmit signals and molecules to other cells. Exosome-mediated signaling contributes to diverse physiological and disease processes, rendering their biogenesis of high biomedical importance. The prevailing hypothesis is that exosomes bud exclusively at endosome membranes and are released only upon endosome fusion with the plasma membrane. Here we tested this hypothesis by examining the intracellular sorting and exosomal secretion of the exosome cargo proteins CD63, CD9, and CD81. We report here that CD9 and CD81 are both localized to the plasma membrane and bud >5-fold more efficiently than endosome-localized CD63. Furthermore, we show that redirecting CD63 from endosomes to the plasma membrane by mutating its endocytosis signal (CD63/Y235A) increased its exosomal secretion ~6-fold, whereas redirecting CD9 to endosomes by adding an endosome targeting signal (CD9/YEVM) reduced its exosomal secretion ~5-fold. These data demonstrate that the plasma membrane is a major site of exosome biogenesis, and more importantly, that cells possess a common pathway for exosome protein budding that operates at both plasma and endosome membranes. Using a combination of single-particle interferometry reflectance (SPIR) imaging and immunofluorescence (IF) microscopy, we also show that variations in exosome composition are controlled by differential intracellular protein trafficking rather than by separate mechanisms of exosome biogenesis. This new view of exosome biogenesis offers a simple explanation for the pronounced compositional heterogeneity of exosomes and a validated roadmap for exosome engineering.SummaryThis study of exosome cargo protein budding reveals that cells use a common pathway for budding exosomes from plasma and endosome membranes, providing a new mechanistic explanation for exosome heterogeneity and a rational roadmap for exosome engineering.

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The Golgi-associated retrograde protein (GARP) complex plays an essential role in the maintenance of the Golgi glycosylation machinery

Molecular Biology of the Cell ◽

10.1091/mbc.e21-04-0169 ◽

2021 ◽

pp. mbc.E21-04-0169

Author(s):

Amrita Khakurel ◽

Tetyana Kudlyk ◽

Juan S. Bonifacino ◽

Vladimir V. Lupashin

Keyword(s):

Steady State ◽

Golgi Complex ◽

Protein Trafficking ◽

Essential Role ◽

Human Cell Lines ◽

Critical Component ◽

Intracellular Protein ◽

The Stability ◽

Endosomal System ◽

Garp Complex

The Golgi complex is a central hub for intracellular protein trafficking and glycosylation. Steady-state localization of glycosylation enzymes is achieved by a combination of mechanisms involving retention and recycling, but the machinery governing these mechanisms is poorly understood. Herein we show that the Golgi-associated retrograde protein (GARP) complex is a critical component of this machinery. Using multiple human cell lines, we show that depletion of GARP subunits impairs Golgi modification of N- and O-glycans, and reduces the stability of glycoproteins and Golgi enzymes. Moreover, GARP-KO cells exhibit reduced retention of glycosylation enzymes in the Golgi. A RUSH assay shows that, in GARP-KO cells, the enzyme beta-1,4-galactosyltransferase 1 is not retained at the Golgi complex but instead is missorted to the endolysosomal system. We propose that the endosomal system is part of the trafficking itinerary of Golgi enzymes or their recycling adaptors and that the GARP complex is essential for recycling and stabilization of the Golgi glycosylation machinery. [Media: see text]

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The adaptor protein AP-4 as a component of the clathrin coat machinery: a morphological study

Biochemical Journal ◽

10.1042/bj20041010 ◽

2005 ◽

Vol 385 (2) ◽

pp. 503-510 ◽

Cited By ~ 28

Author(s):

Nicolas BAROIS ◽

Oddmund BAKKE

Keyword(s):

Golgi Complex ◽

Intracellular Trafficking ◽

Intracellular Localization ◽

Adaptor Protein ◽

Immunogold Labelling ◽

Endocytic Pathway ◽

Transport Vesicles ◽

Cargo Proteins ◽

The Difference ◽

First Time

The four members of the AP (adaptor protein) family are heterotetrameric cytosolic complexes that are involved in the intracellular trafficking of cargo proteins between different organelles. They interact with motifs present in the cytoplasmic tails of their specific cargo proteins at different intracellular locations. While AP-1, AP-2 and AP-3 have been investigated extensively, very few studies have focused on the fourth member, AP-4. In the present study, we report on the intracellular localization of AP-4 in the MDCK (Madin–Darby canine kidney) and MelJuSo cell lines after immunogold labelling of ultrathin cryosections. We find that AP-4 is localized mainly in the Golgi complex, as well as on endosomes and transport vesicles. Interestingly, we show for the first time that AP-4 is localized with the clathrin coat machinery in the Golgi complex and in the endocytic pathway. Furthermore, we find that AP-4 is localized with the CI-MPR (cation-independent mannose 6-phosphate receptor), but not with the transferrin receptor, LAMP-2 (lysosomal-associated membrane protein-2) or invariant chain. The difference in morphology between CI-MPR/AP-4-positive vesicles and CI-MPR/AP-1-positive vesicles raises the possibility that AP-4 acts at a location different from that of AP-1 in the intracellular trafficking pathway of CI-MPR.

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BLOC-1 Interacts with BLOC-2 and the AP-3 Complex to Facilitate Protein Trafficking on Endosomes

Molecular Biology of the Cell ◽

10.1091/mbc.e06-05-0379 ◽

2006 ◽

Vol 17 (9) ◽

pp. 4027-4038 ◽

Cited By ~ 139

Author(s):

Santiago M. Di Pietro ◽

Juan M. Falcón-Pérez ◽

Danièle Tenza ◽

Subba R.G. Setty ◽

Michael S. Marks ◽

...

Keyword(s):

Membrane Protein ◽

Protein Trafficking ◽

Adaptor Protein ◽

Early Endosome ◽

Related Protein ◽

Genes Encoding ◽

Cellular Machinery ◽

Hermansky Pudlak Syndrome ◽

Lysosome Related Organelles ◽

Tyrosinase Related Protein

The adaptor protein (AP)-3 complex is a component of the cellular machinery that controls protein sorting from endosomes to lysosomes and specialized related organelles such as melanosomes. Mutations in an AP-3 subunit underlie a form of Hermansky-Pudlak syndrome (HPS), a disorder characterized by abnormalities in lysosome-related organelles. HPS in humans can also be caused by mutations in genes encoding subunits of three complexes of unclear function, named biogenesis of lysosome-related organelles complex (BLOC)-1, -2, and -3. Here, we report that BLOC-1 interacts physically and functionally with AP-3 to facilitate the trafficking of a known AP-3 cargo, CD63, and of tyrosinase-related protein 1 (Tyrp1), a melanosomal membrane protein previously thought to traffic only independently of AP-3. BLOC-1 also interacts with BLOC-2 to facilitate Tyrp1 trafficking by a mechanism apparently independent of AP-3 function. Both BLOC-1 and -2 localize mainly to early endosome-associated tubules as determined by immunoelectron microscopy. These findings support the idea that BLOC-1 and -2 represent hitherto unknown components of the endosomal protein trafficking machinery.

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