scholarly journals A heterodimeric SNX4­–SNX7 SNX-BAR autophagy complex coordinates ATG9A trafficking for efficient autophagosome assembly

2020 ◽  
Vol 133 (14) ◽  
pp. jcs246306 ◽  
Author(s):  
Zuriñe Antón ◽  
Virginie M. S. Betin ◽  
Boris Simonetti ◽  
Colin J. Traer ◽  
Naomi Attar ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Protein Trafficking ◽  
Mammalian Cells ◽  
Sorting Nexins ◽  
Early Stages ◽  
Peripheral Membrane Proteins ◽  
Golgi Region ◽  
Positive Regulators

ABSTRACTThe sorting nexins (SNXs) are a family of peripheral membrane proteins that direct protein trafficking decisions within the endocytic network. Emerging evidence in yeast and mammalian cells implicates a subgroup of SNXs in selective and non-selective forms of autophagy. Using siRNA and CRISPR-Cas9, we demonstrate that the SNX-BAR protein SNX4 is needed for efficient LC3 (also known as MAP1LC3) lipidation and autophagosome assembly in mammalian cells. SNX-BARs exist as homo- and hetero-dimers, and we show that SNX4 forms functional heterodimers with either SNX7 or SNX30 that associate with tubulovesicular endocytic membranes. Detailed image-based analysis during the early stages of autophagosome assembly reveals that SNX4–SNX7 is an autophagy-specific SNX-BAR heterodimer, required for efficient recruitment and/or retention of core autophagy regulators at the nascent isolation membrane. SNX4 partially colocalises with juxtanuclear ATG9A-positive membranes, with our data linking the autophagy defect upon SNX4 disruption to the mis-trafficking and/or retention of ATG9A in the Golgi region. Taken together, our findings show that the SNX4–SNX7 heterodimer coordinates ATG9A trafficking within the endocytic network to establish productive autophagosome assembly sites, thus extending knowledge of SNXs as positive regulators of autophagy.

scholarly journals A heterodimeric SNX4:SNX7 SNX-BAR autophagy complex coordinates ATG9A trafficking for efficient autophagosome assembly

2020 ◽  
Author(s):  
Zuriñe Antón ◽  
Virginie M.S. Betin ◽  
Boris Simonetti ◽  
Colin J. Traer ◽  
Naomi Attar ◽  
...  
Keyword(s):  
Steady State ◽  
Protein Trafficking ◽  
Mammalian Cells ◽  
Sh3 Domain ◽  
Recycling Endosomes ◽  
Sorting Nexins ◽  
Early Stages ◽  
Domain Type ◽  
Golgi Region ◽  
Summary Statement

ABSTRACTEfficient mammalian autophagosome biogenesis requires coordinated input from other cellular endomembrane compartments. Such coordination includes the stimulated trafficking to autophagosome assembly sites of the essential autophagy proteins, ATG9 and ATG16L1, via distinct endosomal compartments. Protein trafficking within the endocytic network is directed by a conserved family of sorting nexins (SNXs), with previous studies implicating SNX18 (an SH3 domain-type SNX-BAR protein) in the mobilisation of ATG9A and ATG16L1 from recycling endosomes during autophagy. Using siRNA and CRISPR-Cas9, we demonstrate that a second mammalian SNX-BAR, SNX4, is needed for efficient LC3 lipidation and autophagosome assembly in mammalian cells. SNX-BARs exist as homo- and heterodimers, and we show that SNX4 forms functional heterodimers with either SNX7 or SNX30, and that these associate with tubulovesicular endocytic membranes at steady state. Detailed image-based analysis during the early stages of autophagosome assembly reveal that SNX4:SNX7 is the autophagy-specific heterodimeric SNX-BAR complex, required for efficient recruitment/retention of core autophagy regulators at the nascent isolation membrane. SNX4 partially co-localises with juxtanuclear ATG9A-positive membranes, with our data linking the SNX4 autophagy defect to the mis-trafficking and/or retention of ATG9A in the Golgi region. Together, our findings show that the SNX4:SNX7 heterodimer coordinates ATG9A trafficking within the endocytic network to establish productive autophagosome assembly sites.SUMMARY STATEMENTA heterodimeric SNX4:SNX7 SNX-BAR complex regulates mammalian autophagosome assembly through the control of ATG9 trafficking.

scholarly journals Evidence that the entire Golgi apparatus cycles in interphase HeLa cells

2001 ◽  
Vol 155 (4) ◽  
pp. 543-556 ◽  
Author(s):  
Suzanne Miles ◽  
Heather McManus ◽  
Kimberly E. Forsten ◽  
Brian Storrie
Keyword(s):  
Membrane Proteins ◽  
Golgi Apparatus ◽  
Mammalian Cells ◽  
Dynamic Structure ◽  
Dominant Negative ◽  
Integral Membrane Proteins ◽  
Protein Synthesis Inhibitor ◽  
Synthesis Inhibitor ◽  
Golgi Region ◽  
Exit Block

We tested whether the entire Golgi apparatus is a dynamic structure in interphase mammalian cells by assessing the response of 12 different Golgi region proteins to an endoplasmic reticulum (ER) exit block. The proteins chosen spanned the Golgi apparatus and included both Golgi glycosyltransferases and putative matrix proteins. Protein exit from ER was blocked either by microinjection of a GTP-restricted Sar1p mutant protein in the presence of a protein synthesis inhibitor, or by plasmid-encoded expression of the same dominant negative Sar1p. All Golgi region proteins examined lost juxtanuclear Golgi apparatus–like distribution as scored by conventional and confocal fluorescence microscopy in response to an ER exit block, albeit with a differential dependence on Sar1p concentration. Redistribution of GalNAcT2 was more sensitive to low Sar1pdn concentrations than giantin or GM130. Redistribution was most rapid for p27, COPI, and p115. Giantin, GM130, and GalNAcT2 relocated with approximately equal kinetics. Distinct ER accumulation could be demonstrated for all integral membrane proteins. ER-accumulated Golgi region proteins were functional. Photobleaching experiments indicated that Golgi-to-ER protein cycling occurred in the absence of any ER exit block. We conclude that the entire Golgi apparatus is a dynamic structure and suggest that most, if not all, Golgi region–integral membrane proteins cycle through ER in interphase cells.

scholarly journals Dual-colour imaging of membrane protein targeting directed by poa semilatent virus movement protein TGBp3 in plant and mammalian cells

2002 ◽  
Vol 83 (3) ◽  
pp. 651-662 ◽  
Author(s):  
A. A. Zamyatnin ◽  
A. G. Solovyev ◽  
A. A. Sablina ◽  
A. A. Agranovsky ◽  
L. Katul ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Protein Trafficking ◽  
Mammalian Cells ◽  
Protein Targeting ◽  
Fluorescent Protein ◽  
Movement Protein ◽  
Triple Gene Block ◽  
Gene Block ◽  
Movement Function ◽  
Dual Colour

The movement function of poa semilatent hordeivirus (PSLV) is mediated by the triple gene block (TGB) proteins, of which two, TGBp2 and TGBp3, are membrane proteins. TGBp3 is localized to peripheral bodies in the vicinity of the plasma membrane and is able to re-direct TGBp2 from the endoplasmic reticulum (ER) to the peripheral bodies. For imaging of TGBp3-mediated protein targeting, PSLV TGBp3 tagged with a red fluorescent protein (DsRed) was used. Coexpression of DsRed-TGBp3 with GFP targeted to the ER lumen (ER-GFP) demonstrated that ER-GFP was contained in typical ER structures and peripheral bodies formed by TGBp3 protein, suggesting an ER origin for these bodies. In transient coexpression with viral membrane proteins tagged with GFP, DsRed-TGBp3 directed to the peripheral bodies the homologous TGBp2 protein and two unrelated membrane proteins, the 6 kDa movement protein of beet yellows closterovirus and the putative movement protein encoded by the genome component 4 of faba bean necrotic yellows nanovirus. However, coexpression of TGBp3 with GFP derivatives targeted to the ER membranes by artificial hydrophobic tail sequences suggested that targeting to the ER membranes per se was not sufficient for TGBp3-directed protein trafficking to peripheral bodies. TGBp3-induced targeting of TGBp2 also occurred in mammalian cells, indicating the universal nature of the protein trafficking signals and the cotargeting mechanism.

scholarly journals The Golgi S-acylation machinery comprises zDHHC enzymes with major differences in substrate affinity and S-acylation activity

2014 ◽  
Vol 25 (24) ◽  
pp. 3870-3883 ◽  
Author(s):  
Kimon Lemonidis ◽  
Oforiwa A. Gorleku ◽  
Maria C. Sanchez-Perez ◽  
Christopher Grefen ◽  
Luke H. Chamberlain
Keyword(s):  
Membrane Proteins ◽  
Protein Trafficking ◽  
Intracellular Localization ◽  
Substrate Affinity ◽  
Cysteine String Protein ◽  
Peripheral Membrane Proteins ◽  
Study Support ◽  
And Function ◽  
Acylated Proteins ◽  
Selective Interactions

S-acylation, the attachment of fatty acids onto cysteine residues, regulates protein trafficking and function and is mediated by a family of zDHHC enzymes. The S-acylation of peripheral membrane proteins has been proposed to occur at the Golgi, catalyzed by an S-acylation machinery that displays little substrate specificity. To advance understanding of how S-acylation of peripheral membrane proteins is handled by Golgi zDHHC enzymes, we investigated interactions between a subset of four Golgi zDHHC enzymes and two S-acylated proteins—synaptosomal-associated protein 25 (SNAP25) and cysteine-string protein (CSP). Our results uncover major differences in substrate recognition and S-acylation by these zDHHC enzymes. The ankyrin-repeat domains of zDHHC17 and zDHHC13 mediated strong and selective interactions with SNAP25/CSP, whereas binding of zDHHC3 and zDHHC7 to these proteins was barely detectable. Despite this, zDHHC3/zDHHC7 could S-acylate SNAP25/CSP more efficiently than zDHHC17, whereas zDHHC13 lacked S-acylation activity toward these proteins. Overall the results of this study support a model in which dynamic intracellular localization of peripheral membrane proteins is achieved by highly selective recruitment by a subset of zDHHC enzymes at the Golgi, combined with highly efficient S-acylation by other Golgi zDHHC enzymes.

TMCC3 localizes at the three-way junctions for the proper tubular network of the endoplasmic reticulum

2019 ◽  
Vol 476 (21) ◽  
pp. 3241-3260
Author(s):  
Sindhu Wisesa ◽  
Yasunori Yamamoto ◽  
Toshiaki Sakisaka
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Mammalian Cells ◽  
Coiled Coil ◽  
Transmembrane Domains ◽  
Domain Family ◽  
Coiled Coil Domain ◽  
U2os Cells ◽  
Er Membrane

The tubular network of the endoplasmic reticulum (ER) is formed by connecting ER tubules through three-way junctions. Two classes of the conserved ER membrane proteins, atlastins and lunapark, have been shown to reside at the three-way junctions so far and be involved in the generation and stabilization of the three-way junctions. In this study, we report TMCC3 (transmembrane and coiled-coil domain family 3), a member of the TEX28 family, as another ER membrane protein that resides at the three-way junctions in mammalian cells. When the TEX28 family members were transfected into U2OS cells, TMCC3 specifically localized at the three-way junctions in the peripheral ER. TMCC3 bound to atlastins through the C-terminal transmembrane domains. A TMCC3 mutant lacking the N-terminal coiled-coil domain abolished localization to the three-way junctions, suggesting that TMCC3 localized independently of binding to atlastins. TMCC3 knockdown caused a decrease in the number of three-way junctions and expansion of ER sheets, leading to a reduction of the tubular ER network in U2OS cells. The TMCC3 knockdown phenotype was partially rescued by the overexpression of atlastin-2, suggesting that TMCC3 knockdown would decrease the activity of atlastins. These results indicate that TMCC3 localizes at the three-way junctions for the proper tubular ER network.

scholarly journals Basic Residue Clusters in Intrinsically Disordered Regions of Peripheral Membrane Proteins: Modulating 2D Diffusion on Cell Membranes

Physchem ◽  
2021 ◽  
Vol 1 (2) ◽  
pp. 152-162
Author(s):  
Miquel Pons
Keyword(s):  
Membrane Proteins ◽  
Electrostatic Interactions ◽  
Lipid Membrane ◽  
Conformational Ensemble ◽  
Basic Residue ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Peripheral Membrane Proteins ◽  
Charged Residues ◽  
Disordered Regions

A large number of peripheral membrane proteins transiently interact with lipids through a combination of weak interactions. Among them, electrostatic interactions of clusters of positively charged amino acid residues with negatively charged lipids play an important role. Clusters of charged residues are often found in intrinsically disordered protein regions, which are highly abundant in the vicinity of the membrane forming what has been called the disordered boundary of the cell. Beyond contributing to the stability of the lipid-bound state, the pattern of charged residues may encode specific interactions or properties that form the basis of cell signaling. The element of this code may include, among others, the recognition, clustering, and selective release of phosphatidyl inositides, lipid-mediated protein-protein interactions changing the residence time of the peripheral membrane proteins or driving their approximation to integral membrane proteins. Boundary effects include reduction of dimensionality, protein reorientation, biassing of the conformational ensemble of disordered regions or enhanced 2D diffusion in the peri-membrane region enabled by the fuzzy character of the electrostatic interactions with an extended lipid membrane.

A modification of the split-tobacco etch virus method for monitoring interactions between membrane proteins in mammalian cells

2012 ◽  
Vol 423 (1) ◽  
pp. 109-118 ◽  
Author(s):  
Xavier Capdevila-Nortes ◽  
Tania López-Hernández ◽  
Francisco Ciruela ◽  
Raúl Estévez
Keyword(s):  
Membrane Proteins ◽  
Mammalian Cells ◽  
Tobacco Etch Virus

Peripheral Membrane Proteins

1996 ◽  
pp. 355-403 ◽  
Author(s):  
Barbara A. Seaton ◽  
Mary F. Roberts
Keyword(s):  
Membrane Proteins ◽  
Peripheral Membrane Proteins

scholarly journals The intracellular dynamic of protein palmitoylation

2010 ◽  
Vol 191 (7) ◽  
pp. 1229-1238 ◽  
Author(s):  
Christine Salaun ◽  
Jennifer Greaves ◽  
Luke H. Chamberlain
Keyword(s):  
Posttranslational Modifications ◽  
Posttranslational Modification ◽  
Mammalian Cells ◽  
Protein Functionality ◽  
Peripheral Membrane Proteins ◽  
Protein Palmitoylation ◽  
Thioester Bond ◽  
Intracellular Sorting ◽  
Intracellular Dynamic ◽  
Insight Into

S-palmitoylation describes the reversible attachment of fatty acids (predominantly palmitate) onto cysteine residues via a labile thioester bond. This posttranslational modification impacts protein functionality by regulating membrane interactions, intracellular sorting, stability, and membrane micropatterning. Several recent findings have provided a tantalizing insight into the regulation and spatiotemporal dynamics of protein palmitoylation. In mammalian cells, the Golgi has emerged as a possible super-reaction center for the palmitoylation of peripheral membrane proteins, whereas palmitoylation reactions on post-Golgi compartments contribute to the regulation of specific substrates. In addition to palmitoylating and depalmitoylating enzymes, intracellular palmitoylation dynamics may also be controlled through interplay with distinct posttranslational modifications, such as phosphorylation and nitrosylation.

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