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.

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

2020 ◽  
Author(s):  
Rasha Khaddaj ◽  
Muriel Mari ◽  
Stéphanie Cottier ◽  
Fulvio Reggiori ◽  
Roger Schneiter
Keyword(s):  
Endoplasmic Reticulum ◽  
Fusion Proteins ◽  
Mammalian Cells ◽  
Lipid Droplets ◽  
Neutral Lipids ◽  
Bimolecular Fluorescence Complementation ◽  
Steryl Esters ◽  
Perilipin A ◽  
Membrane Spanning ◽  
Mitochondrial Membrane Protein

AbstractLipid droplets (LDs) are globular subcellular structures that mainly serve to store energy in form of neutral lipids, particularly triacylglycerols and steryl esters. LDs are closely associated with the membrane of the endoplasmic reticulum (ER), and are limited by a monolayer membrane of phospholipids 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 spanning proteins could localize to the surface of LDs. To test this, we fused perilipin 3 (PLIN3), a mammalian LD-targeted protein, to ER resident proteins, such as Wbp1 (a N-glycosyl transferase complex subunit), Sec61 (a translocon subunit), and Pmt1 (a protein O-mannosyltransferase). The resulting fusion proteins localize to the periphery of LDs in both yeast and mammalian cells. This peripheral LD localization of the fusion proteins, however, is due to redistribution of the ER around LDs, as revealed by bimolecular fluorescence complementation between ER- and LD-localized partners in cells coexpressing the membrane-anchored perilipin. A LD-tethering function of PLIN3-containing membrane proteins was confirmed by fusing PLIN3 to the cytoplasmic domain of OM14, an outer mitochondrial membrane protein. Expression of OM14-PLIN3 resulted in close apposition of mitochondria and LDs. Taken together, these data indicate that the LD surface constitutes a barrier for ER-localized integral membrane spanning proteins.

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.

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 In Close Proximity: The Lipid Droplet Proteome and Crosstalk With the Endoplasmic Reticulum

Contact ◽  
2018 ◽  
Vol 1 ◽  
pp. 251525641876899 ◽  
Author(s):  
Kirill Bersuker ◽  
James A. Olzmann
Keyword(s):  
Mass Spectrometry ◽  
Endoplasmic Reticulum ◽  
Lipid Droplets ◽  
Neutral Lipids ◽  
26S Proteasome ◽  
High Confidence ◽  
Close Proximity ◽  
Bona Fide ◽  
A Site ◽  
High Degree

Lipid droplets (LDs) are conserved, endoplasmic reticulum (ER)-derived organelles that act as a dynamic cellular repository for neutral lipids. Numerous studies have examined the composition of LD proteomes by using mass spectrometry to identify proteins present in biochemically isolated buoyant fractions that are enriched in LDs. Although many bona fide LD proteins were identified, high levels of non-LD proteins that contaminate buoyant fractions complicate the detection of true LD proteins. To overcome this problem, we recently developed a proximity-labeling proteomic method to define high-confidence LD proteomes. Moreover, employing this approach, we discovered that ER-associated degradation impacts the composition of LD proteomes by targeting select LD proteins for clearance by the 26S proteasome as they transit between the ER and LDs. These findings implicate the ER as a site of LD protein degradation and underscore the high degree of crosstalk between ER and LDs.

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 The yeast lipin orthologue Pah1p is important for biogenesis of lipid droplets

2011 ◽  
Vol 192 (6) ◽  
pp. 1043-1055 ◽  
Author(s):  
Oludotun Adeyo ◽  
Patrick J. Horn ◽  
SungKyung Lee ◽  
Derk D. Binns ◽  
Anita Chandrahas ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Neutral Lipid ◽  
Strong Evidence ◽  
Lipid Droplets ◽  
Neutral Lipids ◽  
Glucose Medium ◽  
Wild Type ◽  
Lipid Levels ◽  
Total Neutral Lipid ◽  
A Site

Lipins are phosphatidate phosphatases that generate diacylglycerol (DAG). In this study, we report that yeast lipin, Pah1p, controls the formation of cytosolic lipid droplets. Disruption of PAH1 resulted in a 63% decrease in droplet number, although total neutral lipid levels did not change. This was accompanied by an accumulation of neutral lipids in the endoplasmic reticulum (ER). The droplet biogenesis defect was not a result of alterations in neutral lipid ratios. No droplets were visible in the absence of both PAH1 and steryl acyltransferases when grown in glucose medium, even though the strain produces as much triacylglycerol as wild type. The requirement of PAH1 for normal droplet formation can be bypassed by a knockout of DGK1. Nem1p, the activator of Pah1p, localizes to a single punctum per cell on the ER that is usually next to a droplet, suggesting that it is a site of droplet assembly. Overall, this study provides strong evidence that DAG generated by Pah1p is important for droplet biogenesis.

scholarly journals Integral Membrane Proteins of the Nuclear Envelope Are Dispersed throughout the Endoplasmic Reticulum during Mitosis

1997 ◽  
Vol 137 (6) ◽  
pp. 1199-1210 ◽  
Author(s):  
Li Yang ◽  
Tinglu Guan ◽  
Larry Gerace
Keyword(s):  
Membrane Proteins ◽  
Nuclear Envelope ◽  
Nuclear Membrane ◽  
Mammalian Cells ◽  
Polyclonal Antibodies ◽  
Integral Membrane Proteins ◽  
Immunogold Electron Microscopy ◽  
Nuclear Pore ◽  
Nuclear Lamins ◽  
Nuclear Membranes

We have analyzed the fate of several integral membrane proteins of the nuclear envelope during mitosis in cultured mammalian cells to determine whether nuclear membrane proteins are present in a vesicle population distinct from bulk ER membranes after mitotic nuclear envelope disassembly or are dispersed throughout the ER. Using immunofluorescence staining and confocal microscopy, we compared the localization of two inner nuclear membrane proteins (laminaassociated polypeptides 1 and 2 [LAP1 and LAP2]) and a nuclear pore membrane protein (gp210) to the distribution of bulk ER membranes, which was determined with lipid dyes (DiOC6 and R6) and polyclonal antibodies. We found that at the resolution of this technique, the three nuclear envelope markers become completely dispersed throughout ER membranes during mitosis. In agreement with these results, we detected LAP1 in most membranes containing ER markers by immunogold electron microscopy of metaphase cells. Together, these findings indicate that nuclear membranes lose their identity as a subcompartment of the ER during mitosis. We found that nuclear lamins begin to reassemble around chromosomes at the end of mitosis at the same time as LAP1 and LAP2 and propose that reassembly of the nuclear envelope at the end of mitosis involves sorting of integral membrane proteins to chromosome surfaces by binding interactions with lamins and chromatin.

scholarly journals A Unique Junctional Interface at Contact Sites Between the Endoplasmic Reticulum and Lipid Droplets

2021 ◽  
Vol 9 ◽  
Author(s):  
Vineet Choudhary ◽  
Roger Schneiter
Keyword(s):  
Endoplasmic Reticulum ◽  
Lipid Droplets ◽  
Neutral Lipids ◽  
Close Contact ◽  
Lipid Storage ◽  
Metabolic Energy ◽  
Lipid Monolayer ◽  
Storage Diseases ◽  
Contact Sites ◽  
Ordered Assembly

Lipid droplets (LDs) constitute compartments dedicated to the storage of metabolic energy in the form of neutral lipids. LDs originate from the endoplasmic reticulum (ER) with which they maintain close contact throughout their life cycle. These ER–LD junctions facilitate the exchange of both proteins and lipids between these two compartments. In recent years, proteins that are important for the proper formation of LDs and localize to ER–LD junctions have been identified. This junction is unique as it is generally believed to invoke a transition from the ER bilayer membrane to a lipid monolayer that delineates LDs. Proper formation of this junction requires the ordered assembly of proteins and lipids at specialized ER subdomains. Without such a well-ordered assembly of LD biogenesis factors, neutral lipids are synthesized throughout the ER membrane, resulting in the formation of aberrant LDs. Such ectopically formed LDs impact ER and lipid homeostasis, resulting in different types of lipid storage diseases. In response to starvation, the ER–LD junction recruits factors that tether the vacuole to these junctions to facilitate LD degradation. In addition, LDs maintain close contacts with peroxisomes and mitochondria for metabolic channeling of the released fatty acids toward beta-oxidation. In this review, we discuss the function of different components that ensure proper functioning of LD contact sites, their role in lipogenesis and lipolysis, and their relation to lipid storage diseases.

scholarly journals Multiple C2 domain-containing transmembrane proteins promote lipid droplet biogenesis and growth at specialized ER subdomains

2021 ◽  
pp. mbc.E20-09-0590
Author(s):  
Amit S. Joshi ◽  
Joey V. Ragusa ◽  
William A. Prinz ◽  
Sarah Cohen
Keyword(s):  
Endoplasmic Reticulum ◽  
Lipid Droplets ◽  
Transmembrane Domain ◽  
Neutral Lipids ◽  
Transmembrane Proteins ◽  
C2 Domain ◽  
General Role ◽  
C2 Domains ◽  
Contact Sites ◽  
Er Membrane

Lipid droplets (LDs) are neutral lipid-containing organelles enclosed in a single monolayer of phospholipids. LD formation begins with the accumulation of neutral lipids within the bilayer of the endoplasmic reticulum (ER) membrane. It is not known how the sites of formation of nascent LDs in the ER membrane are determined. Here we show that multiple C2 domain-containing transmembrane proteins, MCTP1 and MCTP2, are at sites of LD formation in specialized ER subdomains. We show that the transmembrane domain (TMD) of these proteins is similar to a reticulon homology domain. Like reticulons, these proteins tubulate the ER membrane and favor highly curved regions of the ER. Our data indicate that the MCTP TMDs promote LD biogenesis, increasing LD number. MCTPs co-localize with seipin, a protein involved in LD biogenesis, but form more stable microdomains in the ER. The MCTP C2 domains bind charged lipids and regulate LD size, likely by mediating ER-LD contact sites. Together, our data indicate that MCTPs form microdomains within ER tubules that regulate LD biogenesis, size, and ER-LD contacts. Interestingly, MCTP punctae colocalized with other organelles as well, suggesting that these proteins may play a more general role in linking tubular ER to organelle contact sites. [Media: see text] [Media: see text]

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