Control of endoplasmic reticulum membrane biogenesis by regulators of lipid metabolism

ABSTRACTCells dynamically adapt organelle size to current physiological demand. Organelle growth and proliferation require membrane biogenesis and need to be coordinated with lipid metabolism. The endoplasmic reticulum (ER) can undergo massive expansion but the mechanisms that govern ER membrane biogenesis are unclear. Here, we genetically screen for factors mediating ER expansion in budding yeast and identify lipid synthesis enzymes and the ER transmembrane protein Ice2 as strong hits. Ice2 inhibits the conserved phosphatidic acid phosphatase Pah1 by opposing the activity of the Nem1-Spo7 complex. This regulation counteracts the production of storage lipids and directs lipid metabolism towards membrane biogenesis. Furthermore, Ice2 acts in concert with the transcriptional control of lipid synthesis enzymes and cooperates with the unfolded protein response to maintain ER homeostasis. These findings establish the regulation of the lipin ortholog Pah1 as a key determinant of ER membrane biogenesis.

Download Full-text

The unfolded protein response coordinates the production of endoplasmic reticulum protein and endoplasmic reticulum membrane.

Molecular Biology of the Cell ◽

10.1091/mbc.8.9.1805 ◽

1997 ◽

Vol 8 (9) ◽

pp. 1805-1814 ◽

Cited By ~ 277

Author(s):

J S Cox ◽

R E Chapman ◽

P Walter

Keyword(s):

Endoplasmic Reticulum ◽

Unfolded Protein Response ◽

Secretory Pathway ◽

Lipid Synthesis ◽

Secretory Proteins ◽

Endoplasmic Reticulum Membrane ◽

Yeast Saccharomyces Cerevisiae ◽

Unfolded Protein ◽

The Unfolded Protein Response ◽

Protein Response

The endoplasmic reticulum (ER) is a multifunctional organelle responsible for production of both lumenal and membrane components of secretory pathway compartments. Secretory proteins are folded, processed, and sorted in the ER lumen and lipid synthesis occurs on the ER membrane itself. In the yeast Saccharomyces cerevisiae, synthesis of ER components is highly regulated: the ER-resident proteins by the unfolded protein response and membrane lipid synthesis by the inositol response. We demonstrate that these two responses are intimately linked, forming different branches of the same pathway. Furthermore, we present evidence indicating that this coordinate regulation plays a role in ER biogenesis.

Download Full-text

The beta subunit of the signal recognition particle receptor is a transmembrane GTPase that anchors the alpha subunit, a peripheral membrane GTPase, to the endoplasmic reticulum membrane.

The Journal of Cell Biology ◽

10.1083/jcb.128.3.273 ◽

1995 ◽

Vol 128 (3) ◽

pp. 273-282 ◽

Cited By ~ 76

Author(s):

J D Miller ◽

S Tajima ◽

L Lauffer ◽

P Walter

Keyword(s):

Endoplasmic Reticulum ◽

Protein Targeting ◽

Signal Sequence ◽

Transmembrane Protein ◽

Signal Recognition Particle ◽

Endoplasmic Reticulum Membrane ◽

Protein Chain ◽

Signal Recognition ◽

Signal Recognition Particle Receptor ◽

Er Membrane

The signal recognition particle receptor (SR) is required for the cotranslational targeting of both secretory and membrane proteins to the endoplasmic reticulum (ER) membrane. During targeting, the SR interacts with the signal recognition particle (SRP) which is bound to the signal sequence of the nascent protein chain. This interaction catalyzes the GTP-dependent transfer of the nascent chain from SRP to the protein translocation apparatus in the ER membrane. The SR is a heterodimeric protein comprised of a 69-kD subunit (SR alpha) and a 30-kD subunit (SR beta) which are associated with the ER membrane in an unknown manner. SR alpha and the 54-kD subunits of SRP (SRP54) each contain related GTPase domains which are required for SR and SRP function. Molecular cloning and sequencing of a cDNA encoding SR beta revealed that SR beta is a transmembrane protein and, like SR alpha and SRP54, is a member of the GTPase superfamily. Although SR beta defines its own GTPase subfamily, it is distantly related to ARF and Sar1. Using UV cross-linking, we confirm that SR beta binds GTP specifically. Proteolytic digestion experiments show that SR alpha is required for the interaction of SRP with SR. SR alpha appears to be peripherally associated with the ER membrane, and we suggest that SR beta, as an integral membrane protein, mediates the membrane association of SR alpha. The discovery of its guanine nucleotide-binding domain, however, makes it likely that its role is more complex than that of a passive anchor for SR alpha. These findings suggest that a cascade of three directly interacting GTPases functions during protein targeting to the ER membrane.

Download Full-text

An essential dimer-forming subregion of the endoplasmic reticulum stress sensor Ire1

Biochemical Journal ◽

10.1042/bj20050640 ◽

2005 ◽

Vol 391 (1) ◽

pp. 135-142 ◽

Cited By ~ 27

Author(s):

Daisuke Oikawa ◽

Yukio Kimata ◽

Masato Takeuchi ◽

Kenji Kohno

Keyword(s):

Endoplasmic Reticulum ◽

Recombinant Protein ◽

Endoplasmic Reticulum Stress ◽

Transmembrane Protein ◽

Type I ◽

Unfolded Protein ◽

The Unfolded Protein Response ◽

Protein Response ◽

Recombinant Fragments ◽

Made In

The luminal domain of the type I transmembrane protein Ire1 senses endoplasmic reticulum stress by an undefined mechanism to up-regulate the signalling pathway for the unfolded protein response. Previously, we proposed that the luminal domain of yeast Ire1 is divided into five subregions, termed subregions I–V sequentially from the N-terminus. Ire1 lost activity when internal deletions of subregion II or IV were made. In the present paper, we show that partial proteolysis of a recombinant protein consisting of the Ire1 luminal domain suggests that subregions II–IV are tightly folded. We also show that a recombinant protein of subregions II–IV formed homodimers, and that this homodimer formation was impaired by an internal deletion of subregion IV. Furthermore, recombinant fragments of subregion IV exhibited a self-binding ability. Therefore, although its sequence is little conserved evolutionarily, subregion IV plays an essential role to promote Ire1 dimer formation.

Download Full-text

Autophagosome formation in relation to the endoplasmic reticulum

Journal of Biomedical Science ◽

10.1186/s12929-020-00691-6 ◽

2020 ◽

Vol 27 (1) ◽

Author(s):

Yo-hei Yamamoto ◽

Takeshi Noda

Keyword(s):

Endoplasmic Reticulum ◽

Membrane Protein ◽

De Novo ◽

Transmembrane Protein ◽

Lipid Transfer ◽

Membrane Structures ◽

Autophagosome Formation ◽

Selective Autophagy ◽

The Relationship ◽

Er Membrane

Abstract Autophagy is a process in which a myriad membrane structures called autophagosomes are formed de novo in a single cell, which deliver the engulfed substrates into lysosomes for degradation. The size of the autophagosomes is relatively uniform in non-selective autophagy and variable in selective autophagy. It has been recently established that autophagosome formation occurs near the endoplasmic reticulum (ER). In this review, we have discussed recent advances in the relationship between autophagosome formation and endoplasmic reticulum. Autophagosome formation occurs near the ER subdomain enriched with phospholipid synthesizing enzymes like phosphatidylinositol synthase (PIS)/CDP-diacylglycerol-inositol 3-phosphatidyltransferase (CDIPT) and choline/ethanolamine phosphotransferase 1 (CEPT1). Autophagy-related protein 2 (Atg2), which is involved in autophagosome formation has a lipid transfer capacity and is proposed to directly transfer the lipid molecules from the ER to form autophagosomes. Vacuole membrane protein 1 (VMP1) and transmembrane protein 41b (TMEM41b) are ER membrane proteins that are associated with the formation of the subdomain. Recently, we have reported that an uncharacterized ER membrane protein possessing the DNAJ domain, called ERdj8/DNAJC16, is associated with the regulation of the size of autophagosomes. The localization of ERdj8/DNAJC16 partially overlaps with the PIS-enriched ER subdomain, thereby implying its association with autophagosome size determination.

Download Full-text

A conserved family of proteins facilitates nascent lipid droplet budding from the ER

The Journal of Cell Biology ◽

10.1083/jcb.201505067 ◽

2015 ◽

Vol 211 (2) ◽

pp. 261-271 ◽

Cited By ~ 137

Author(s):

Vineet Choudhary ◽

Namrata Ojha ◽

Andy Golden ◽

William A. Prinz

Keyword(s):

Electron Microscopy ◽

Endoplasmic Reticulum ◽

Lipid Metabolism ◽

Caenorhabditis Elegans ◽

Lipid Droplet ◽

Lipid Droplets ◽

De Novo ◽

Fat Storage ◽

Higher Eukaryotes ◽

Er Membrane

Lipid droplets (LDs) are found in all cells and play critical roles in lipid metabolism. De novo LD biogenesis occurs in the endoplasmic reticulum (ER) but is not well understood. We imaged early stages of LD biogenesis using electron microscopy and found that nascent LDs form lens-like structures that are in the ER membrane, raising the question of how these nascent LDs bud from the ER as they grow. We found that a conserved family of proteins, fat storage-inducing transmembrane (FIT) proteins, is required for proper budding of LDs from the ER. Elimination or reduction of FIT proteins in yeast and higher eukaryotes causes LDs to remain in the ER membrane. Deletion of the single FIT protein in Caenorhabditis elegans is lethal, suggesting that LD budding is an essential process in this organism. Our findings indicated that FIT proteins are necessary to promote budding of nascent LDs from the ER.

Download Full-text

TheSaccharomyces cerevisiaePrenylcysteine Carboxyl Methyltransferase Ste14p Is in the Endoplasmic Reticulum Membrane

Molecular Biology of the Cell ◽

10.1091/mbc.9.8.2231 ◽

1998 ◽

Vol 9 (8) ◽

pp. 2231-2247 ◽

Cited By ~ 80

Author(s):

Julia D. Romano ◽

Walter K. Schmidt ◽

Susan Michaelis

Keyword(s):

Endoplasmic Reticulum ◽

Subcellular Fractionation ◽

Endoplasmic Reticulum Membrane ◽

Carboxyl Methylation ◽

C Terminus ◽

Eukaryotic Proteins ◽

Internal Site ◽

Membrane Spanning ◽

Er Membrane

Eukaryotic proteins containing a C-terminal CAAX motif undergo a series of posttranslational CAAX-processing events that include isoprenylation, C-terminal proteolytic cleavage, and carboxyl methylation. We demonstrated previously that the STE14gene product of Saccharomyces cerevisiae mediates the carboxyl methylation step of CAAX processing in yeast. In this study, we have investigated the subcellular localization of Ste14p, a predicted membrane-spanning protein, using a polyclonal antibody generated against the C terminus of Ste14p and an in vitro methyltransferase assay. We demonstrate by immunofluorescence and subcellular fractionation that Ste14p and its associated activity are localized to the endoplasmic reticulum (ER) membrane of yeast. In addition, other studies from our laboratory have shown that the CAAX proteases are also ER membrane proteins. Together these results indicate that the intracellular site of CAAX protein processing is the ER membrane, presumably on its cytosolic face. Interestingly, the insertion of a hemagglutinin epitope tag at the N terminus, at the C terminus, or at an internal site disrupts the ER localization of Ste14p and results in its mislocalization, apparently to the Golgi. We have also expressed the Ste14p homologue from Schizosaccharomyces pombe, mam4p, in S. cerevisiae and have shown that mam4p complements a Δste14 mutant. This finding, plus additional recent examples of cross-species complementation, indicates that the CAAX methyltransferase family consists of functional homologues.

Download Full-text

Endoplasmic Reticulum Stress and Lipid Metabolism: Mechanisms and Therapeutic Potential

Biochemistry Research International ◽

10.1155/2012/841362 ◽

2012 ◽

Vol 2012 ◽

pp. 1-13 ◽

Cited By ~ 95

Author(s):

Sana Basseri ◽

Richard C. Austin

Keyword(s):

Endoplasmic Reticulum ◽

Protein Folding ◽

Lipid Metabolism ◽

Er Stress ◽

Therapeutic Potential ◽

Critical Role ◽

Signal Transduction Pathway ◽

Unfolded Protein ◽

The Unfolded Protein Response ◽

Stress Dependent

The endoplasmic reticulum (ER) plays a crucial role in protein folding, assembly, and secretion. Disruption of ER homeostasis may lead to accumulation of misfolded or unfolded proteins in the ER lumen, a condition referred to as ER stress. In response to ER stress, a signal transduction pathway known as the unfolded protein response (UPR) is activated. UPR activation allows the cell to cope with an increased protein-folding demand on the ER. Recent studies have shown that ER stress/UPR activation plays a critical role in lipid metabolism and homeostasis. ER-stress-dependent dysregulation of lipid metabolism may lead to dyslipidemia, insulin resistance, cardiovascular disease, type 2 diabetes, and obesity. In this paper, we examine recent findings illustrating the important role ER stress/UPR signalling pathways play in regulation of lipid metabolism, and how they may lead to dysregulation of lipid homeostasis.

Download Full-text

Different subcellular localization of Saccharomyces cerevisiae HMG-CoA reductase isozymes at elevated levels corresponds to distinct endoplasmic reticulum membrane proliferations.

Molecular Biology of the Cell ◽

10.1091/mbc.7.5.769 ◽

1996 ◽

Vol 7 (5) ◽

pp. 769-789 ◽

Cited By ~ 81

Author(s):

A J Koning ◽

C J Roberts ◽

R L Wright

Keyword(s):

Saccharomyces Cerevisiae ◽

Endoplasmic Reticulum ◽

Subcellular Localization ◽

Nuclear Envelope ◽

Fluorescent Protein ◽

Endoplasmic Reticulum Membrane ◽

Hmg Coa Reductase ◽

Endogenous Expression ◽

Coa Reductase ◽

Er Membrane

In all eucaryotic cell types analyzed, proliferations of the endoplasmic reticulum (ER) can be induced by increasing the levels of certain integral ER proteins. One of the best characterized of these proteins is HMG-CoA reductase, which catalyzes the rate-limiting step in sterol biosynthesis. We have investigated the subcellular distributions of the two HMG-CoA reductase isozymes in Saccharomyces cerevisiae and the types of ER proliferations that arise in response to elevated levels of each isozyme. At endogenous expression levels, Hmg1p and Hmg2p were both primarily localized in the nuclear envelope. However, at increased levels, the isozymes displayed distinct subcellular localization patterns in which each isozyme was predominantly localized in a different region of the ER. Specifically, increased levels of Hmg1p were concentrated in the nuclear envelope, whereas increased levels of Hmg2p were concentrated in the peripheral ER. In addition, an Hmg2p chimeric protein containing a 77-amino acid lumenal segment from Hmg1p was localized in a pattern that resembled that of Hmg1p when expressed at increased levels. Reflecting their different subcellular distributions, elevated levels of Hmg1p and Hmg2p induced sets of ER membrane proliferations with distinct morphologies. The ER membrane protein, Sec61p, was localized in the membranes induced by both Hmg1p and Hmg2p green fluorescent protein (GFP) fusions. In contrast, the lumenal ER protein, Kar2p, was present in Hmg1p:GFP membranes, but only rarely in Hmg2p:GFP membranes. These results indicated that the membranes synthesized in response to Hmg1p and Hmg2p were derived from the ER, but that the membranes were not identical in protein composition. We determined that the different types of ER proliferations were not simply due to quantitative differences in protein amounts or to the different half-lives of the two isozymes. It is possible that the specific distributions of the two yeast HMG-CoA reductase isozymes and their corresponding membrane proliferations may reveal regions of the ER that are specialized for certain branches of the sterol biosynthetic pathway.

Download Full-text

A role for BiP as an adjustor for the endoplasmic reticulum stress-sensing protein Ire1

The Journal of Cell Biology ◽

10.1083/jcb.200405153 ◽

2004 ◽

Vol 167 (3) ◽

pp. 445-456 ◽

Cited By ~ 175

Author(s):

Yukio Kimata ◽

Daisuke Oikawa ◽

Yusuke Shimizu ◽

Yuki Ishiwata-Kimata ◽

Kenji Kohno

Keyword(s):

Endoplasmic Reticulum ◽

Er Stress ◽

Binding Site ◽

Transmembrane Domain ◽

Transmembrane Protein ◽

Sensory System ◽

Type I ◽

Stress Signal ◽

Stress Sensing ◽

The Unfolded Protein Response

In the unfolded protein response, the type I transmembrane protein Ire1 transmits an endoplasmic reticulum (ER) stress signal to the cytoplasm. We previously reported that under nonstressed conditions, the ER chaperone BiP binds and represses Ire1. It is still unclear how this event contributes to the overall regulation of Ire1. The present Ire1 mutation study shows that the luminal domain possesses two subregions that seem indispensable for activity. The BiP-binding site was assigned not to these subregions, but to a region neighboring the transmembrane domain. Phenotypic comparison of several Ire1 mutants carrying deletions in the indispensable subregions suggests these subregions are responsible for multiple events that are prerequisites for activation of the overall Ire1 proteins. Unexpectedly, deletion of the BiP-binding site rendered Ire1 unaltered in ER stress inducibility, but hypersensitive to ethanol and high temperature. We conclude that in the ER stress-sensory system BiP is not the principal determinant of Ire1 activity, but an adjustor for sensitivity to various stresses.

Download Full-text

RNF-121 Is an Endoplasmic Reticulum-Membrane E3 Ubiquitin Ligase Involved in the Regulation of β-Integrin

Molecular Biology of the Cell ◽

10.1091/mbc.e09-09-0774 ◽

2010 ◽

Vol 21 (11) ◽

pp. 1788-1798 ◽

Cited By ~ 26

Author(s):

Amir Darom ◽

Ulrike Bening-Abu-Shach ◽

Limor Broday

Keyword(s):

Endoplasmic Reticulum ◽

Ubiquitin Ligase ◽

Gonad Development ◽

Ring Finger ◽

Endoplasmic Reticulum Membrane ◽

Integrin Receptors ◽

Germline Development ◽

Unfolded Protein ◽

Regulator Protein ◽

The Unfolded Protein Response

We report on the characterization of RNF-121, an evolutionarily conserved E3 ligase RING finger protein that is expressed in the endoplasmic reticulum (ER) of various cells and tissues in Caenorhabditis elegans . Inactivation of RNF-121 induced an elevation in BiP expression and increased the sensitivity of worms to ER stress. Genetic analysis placed RNF-121 downstream of the unfolded protein response (UPR) regulator protein kinase-like endoplasmic reticulum kinase (PERK). We identify PAT-3::GFP, the β subunit of the heterodimeric integrin receptors, as an RNF-121 substrate; whereas induction of RNF-121 expression reduced the level of PAT-3::GFP in the gonad distal tip cells, inhibition of RNF-121 led to the accumulation of stably bound PAT-3::GFP inclusions. Correspondingly, overexpression of RNF-121 during early stages of gonad development led to aberrations in germline development and gonad migration that overlap with those observed after PAT-3 inactivation. The formation of these gonad abnormalities required functional ER-associated degradation (ERAD) machinery. Our findings identify RNF-121 as an ER-anchored ubiquitin ligase that plays a specific role in the ERAD pathway by linking it to the regulation of the cell adhesion integrin receptors.

Download Full-text