scholarly journals Characterization of constitutive ER-phagy of excess membrane proteins

PLoS Genetics ◽  
2020 ◽  
Vol 16 (12) ◽  
pp. e1009255
Author(s):  
Zhanna Lipatova ◽  
Valeriya Gyurkovska ◽  
Sarah F. Zhao ◽  
Nava Segev
Keyword(s):  
Quality Control ◽  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Human Health ◽  
Mammalian Cells ◽  
Normal Growth ◽  
Integral Membrane Proteins ◽  
Yeast Cells ◽  
Cellular Proteins

Thirty percent of all cellular proteins are inserted into the endoplasmic reticulum (ER), which spans throughout the cytoplasm. Two well-established stress-induced pathways ensure quality control (QC) at the ER: ER-phagy and ER-associated degradation (ERAD), which shuttle cargo for degradation to the lysosome and proteasome, respectively. In contrast, not much is known about constitutive ER-phagy. We have previously reported that excess of integral-membrane proteins is delivered from the ER to the lysosome via autophagy during normal growth of yeast cells. Whereas endogenously expressed ER resident proteins serve as cargos at a basal level, this level can be induced by overexpression of membrane proteins that are not ER residents. Here, we characterize this pathway as constitutive ER-phagy. Constitutive and stress-induced ER-phagy share the basic macro-autophagy machinery including the conserved Atgs and Ypt1 GTPase. However, induction of stress-induced autophagy is not needed for constitutive ER-phagy to occur. Moreover, the selective receptors needed for starvation-induced ER-phagy, Atg39 and Atg40, are not required for constitutive ER-phagy and neither these receptors nor their cargos are delivered through it to the vacuole. As for ERAD, while constitutive ER-phagy recognizes cargo different from that recognized by ERAD, these two ER-QC pathways can partially substitute for each other. Because accumulation of membrane proteins is associated with disease, and constitutive ER-phagy players are conserved from yeast to mammalian cells, this process could be critical for human health.

scholarly journals Characterization of Constitutive macro-ER-phagy

2020 ◽  
Author(s):  
Zhanna Lipatova ◽  
Valeriya Gyurkovska ◽  
Sarah F. Zhao ◽  
Nava Segev
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Er Stress ◽  
Mammalian Cells ◽  
Normal Growth ◽  
Integral Membrane Proteins ◽  
Yeast Cells ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response

AbstractThirty percent of all cellular proteins are inserted into the endoplasmic reticulum (ER), which spans throughout the cytoplasm. Two well-established stress-induced pathways ensure quality control (QC) at the ER: ER-phagy and ER-associated degradation (ERAD), which shuttle cargo for degradation to the lysosome and proteasome, respectively. In contrast, not much is known about constitutive ER-phagy. We have previously reported that excess of integral-membrane proteins is delivered from the ER to the lysosome via autophagy during normal growth of yeast cells. Here, we characterize this pathway as constitutive ER-phagy. Constitutive and stress-induced ER-phagy share the basic macro-autophagy machinery including the conserved Atgs and Ypt1 GTPase. However, induction of stress-induced autophagy is not needed for constitutive ER-phagy to occur. Moreover, the selective receptors needed for starvation-induced ER-phagy, Atg39 and Atg40, are not required for constitutive ER-phagy and neither these receptors nor their cargos are delivered through it to the vacuole. As for ERAD, while constitutive ER-phagy recognizes cargo different from that recognized by ERAD, these two ER-QC pathways can partially substitute for each other. Because accumulation of membrane proteins is associated with disease, and constitutive ER-phagy players are conserved from yeast to mammalian cells, this process could be critical for human health.Author SummaryAccumulation of excess proteins can lead to their aggregation, which in turn can cause multiple disorders, notably neurodegenerative disease. Nutritional and endoplasmic-reticulum (ER) stress stimulate autophagy and ER-associated degradation (ERAD) to clear excess and misfolded proteins, respectively. However, not much is known about clearance of excess proteins during normal growth. We have previously shown that excess integral-membrane proteins are cleared from the ER by macro-autophagy during normal growth of yeast cells. Here we characterize this pathway as constitutive ER-phagy. While this pathway shares machinery of core Atgs and autophagosomes with nutritional stress-induced ER-phagy, it differs from the latter: It is independent of the stress response and of receptors needed for stress-induced ER-phagy, and while stress-induced ER-phagy is not discriminatory, constitutive ER-phagy has specific cargos. However, when constitutive ER-phagy fails, machinery specific to stress-induced ER-phagy can process its cargo. Moreover, constitutive ER-phagy is also not dependent on ER-stress or the unfolded protein response (UPR) stimulated by this stress, although its failure elicits UPR. Finally, constitutive ER-phagy and ERAD can partially process each other’s cargo upon failure. In summary, constitutive ER-phagy, which clears excess integral-membrane proteins from the ER during normal growth does not require nutritional or ER stress for its function.

scholarly journals The surface of lipid droplets constitutes a barrier for endoplasmic reticulum residential integral membrane proteins

2021 ◽  
Author(s):  
Rasha Khaddaj ◽  
Muriel Mari ◽  
Stéphanie Cottier ◽  
Fulvio Reggiori ◽  
Roger Schneiter
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Fusion Proteins ◽  
Mammalian Cells ◽  
Lipid Droplets ◽  
Neutral Lipids ◽  
Integral Membrane Proteins ◽  
Bimolecular Fluorescence Complementation ◽  
Amphipathic Helix ◽  
Mitochondrial Membrane Protein

Lipid droplets (LDs) are globular subcellular structures that store neutral lipids. LDs are closely associated with the endoplasmic reticulum (ER), and are limited by a phospholipid monolayer harboring a specific set of proteins. Most of these proteins associate with LDs through either an amphipathic helix or a membrane-embedded hairpin motif. Here we address the question whether integral membrane proteins could localize to the surface of LDs. To test this, we fused perilipin 3 (PLIN3), a mammalian LD-targeted protein, to ER residential proteins. The resulting fusion proteins localized to the periphery of LDs in both yeast and mammalian cells. This peripheral LD localization of the fusion proteins, however, was due to a redistribution of the ER around LDs, as revealed by bimolecular fluorescence complementation between ER- and LD-localized partners. A LD-tethering function of PLIN3-containing membrane proteins was confirmed by fusing PLIN3 to the cytoplasmic domain of an outer mitochondrial membrane protein, OM14. Expression of OM14-PLIN3 induced a close apposition between LDs and mitochondria. These data indicate that the ER-LD junction constitutes a barrier for ER-residential integral membrane proteins.

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 Septin-dependent compartmentalization of the endoplasmic reticulum during yeast polarized growth

2005 ◽  
Vol 169 (6) ◽  
pp. 897-908 ◽  
Author(s):  
Cosima Luedeke ◽  
Stéphanie Buvelot Frei ◽  
Ivo Sbalzarini ◽  
Heinz Schwarz ◽  
Anne Spang ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Membrane Proteins ◽  
Diffusion Barriers ◽  
Yeast Cells ◽  
Dependent Manner ◽  
Protein Diffusion ◽  
Ultrastructural Studies ◽  
Bud Neck ◽  
Er Membrane

Polarized cells frequently use diffusion barriers to separate plasma membrane domains. It is unknown whether diffusion barriers also compartmentalize intracellular organelles. We used photobleaching techniques to characterize protein diffusion in the yeast endoplasmic reticulum (ER). Although a soluble protein diffused rapidly throughout the ER lumen, diffusion of ER membrane proteins was restricted at the bud neck. Ultrastructural studies and fluorescence microscopy revealed the presence of a ring of smooth ER at the bud neck. This ER domain and the restriction of diffusion for ER membrane proteins through the bud neck depended on septin function. The membrane-associated protein Bud6 localized to the bud neck in a septin-dependent manner and was required to restrict the diffusion of ER membrane proteins. Our results indicate that Bud6 acts downstream of septins to assemble a fence in the ER membrane at the bud neck. Thus, in polarized yeast cells, diffusion barriers compartmentalize the ER and the plasma membrane along parallel lines.

Characterization of two integral membrane proteins located in the protein bodies of pumpkin seeds

1995 ◽  
Vol 28 (6) ◽  
pp. 1089-1101 ◽  
Author(s):  
Kaori Inoue ◽  
Yuka Takeuchi ◽  
Mikio Nishimura ◽  
Ikuko Hara-Nishimura
Keyword(s):  
Membrane Proteins ◽  
Integral Membrane Proteins ◽  
Protein Bodies ◽  
Pumpkin Seeds

Characterization of a polymorphic family of integral membrane proteins in promastigotes of different Leishmania species

1994 ◽  
Vol 67 (1) ◽  
pp. 103-113 ◽  
Author(s):  
Fiona M. Symons ◽  
Peter J. Murray ◽  
Hong Ji ◽  
Richard J. Simpson ◽  
Amelia H. Osborn ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Leishmania Species ◽  
Integral Membrane Proteins

Solubilization, Purification, and Characterization of Integral Membrane Proteins

2011 ◽  
pp. 317-360 ◽  
Author(s):  
Víctor Lórenz-Fonfría ◽  
Alex Perálvarez-Marín ◽  
Esteve Padrós ◽  
Tzvetana Lazarova
Keyword(s):  
Membrane Proteins ◽  
Integral Membrane Proteins ◽  
Purification And Characterization

Identification and functional characterization of a novel human protein highly related to the yeast dynamin-like GTPase Vps1p

1998 ◽  
Vol 111 (10) ◽  
pp. 1341-1349 ◽  
Author(s):  
M. Imoto ◽  
I. Tachibana ◽  
R. Urrutia
Keyword(s):  
Endoplasmic Reticulum ◽  
Cho Cells ◽  
Mammalian Cells ◽  
Functional Characterization ◽  
Luciferase Reporter ◽  
Pleckstrin Homology ◽  
Rich Region ◽  
Gtpase Domain ◽  
Proline Rich Region

Dynamin proteins containing a GTPase domain, a pleckstrin homology motif and a proline-rich tail participate in receptor-mediated endocytosis in organisms ranging from insects to vertebrates. In addition, dynamin-related GTPases, such as the yeast Golgi protein Vps1p, which lack both the pleckstrin homology motif and the proline-rich region, participate in vesicular transport within the secretory pathway in lower eukaryotes. However, no data is available on the existence of Vps1p-like proteins in mammalian cells. In this study, we report the identification and characterization of a novel gene encoding a human dynamin-related protein, DRP1, displaying high similarity to the Golgi dynamin-like protein Vps1p from yeast and to a Caenorhabditis elegans protein deposited in the databank. These proteins are highly conserved in their N-terminal tripartite GTPase domain but lack the pleckstrin homology motif and proline-rich region. Northern blot analysis reveals that the DRP1 mRNA is detected at high levels in human muscle, heart, kidney and brain. Immunolocalization studies in Chinese hamster ovary (CHO) cells using an epitope-tagged form of DRP1 and confocal microscopy show that this protein is concentrated in a perinuclear region that labels with the endoplasmic reticulum marker DiOC6(3) and the Golgi marker C5-DMB-Cer. In addition, the localization of DRP1 is highly similar to the localization of the endoplasmic reticulum and cis-Golgi GTPase Rab1A, but not to the staining for the trans-Golgi GTPase Rab6. Furthermore, overexpression of a cDNA encoding a GTP binding site mutant of DRP1 (DRP1(K38E)) in CHO cells decreases the amount of a secreted luciferase reporter protein, whereas the overexpression of wild-type DRP1 increases the secretion of this marker. Together, these results constitute the first structural and functional characterization of a mammalian protein similar to the yeast dynamin-related GTPase Vps1p and indicate that the participation of these proteins in secretion has been conserved throughout evolution.

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.

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