Protein translocation mutants defective in the insertion of integral membrane proteins into the endoplasmic reticulum

The rough endoplasmic reticulum (r.e.r.) has been postulated to possess a single translation-coupled translocation system (in multiple copies) that effects signal sequence-mediated translocation of all secretory and lysosomal proteins and integration of all integral membrane proteins whose port of entry is the rough endoplasmic reticulum (G. Blobel 1980 Proc. natn. Acad. Sci. U.S.A. 77, 1496—1500). Two proteins have been isolated that are components of the r.e.r. translocation system. Their properties and function in protein translocation across and integration into membranes are discussed.

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Characterization of constitutive ER-phagy of excess membrane proteins

PLoS Genetics ◽

10.1371/journal.pgen.1009255 ◽

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.

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Structure of the posttranslational Sec protein-translocation channel complex from yeast

Science ◽

10.1126/science.aav6740 ◽

2018 ◽

Vol 363 (6422) ◽

pp. 84-87 ◽

Cited By ~ 32

Author(s):

Samuel Itskanov ◽

Eunyong Park

Keyword(s):

Electron Microscopy ◽

Saccharomyces Cerevisiae ◽

Endoplasmic Reticulum ◽

Membrane Proteins ◽

Binding Sites ◽

Protein Translocation ◽

Secretory Proteins ◽

Conducting Channel ◽

Cryo Electron Microscopy ◽

Lateral Gate

The Sec61 protein-conducting channel mediates transport of many proteins, such as secretory proteins, across the endoplasmic reticulum (ER) membrane during or after translation. Posttranslational transport is enabled by two additional membrane proteins associated with the channel, Sec63 and Sec62, but its mechanism is poorly understood. We determined a structure of the Sec complex (Sec61-Sec63-Sec71-Sec72) from Saccharomyces cerevisiae by cryo–electron microscopy (cryo-EM). The structure shows that Sec63 tightly associates with Sec61 through interactions in cytosolic, transmembrane, and ER-luminal domains, prying open Sec61’s lateral gate and translocation pore and thus activating the channel for substrate engagement. Furthermore, Sec63 optimally positions binding sites for cytosolic and luminal chaperones in the complex to enable efficient polypeptide translocation. Our study provides mechanistic insights into eukaryotic posttranslational protein translocation.

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Biogenesis of tail-anchored proteins

Biochemical Society Transactions ◽

10.1042/bst0311238 ◽

2003 ◽

Vol 31 (6) ◽

pp. 1238-1242 ◽

Cited By ~ 31

Author(s):

N. Borgese ◽

S. Brambillasca ◽

P. Soffientini ◽

M. Yabal ◽

M. Makarow

Keyword(s):

Amino Acids ◽

Endoplasmic Reticulum ◽

Membrane Proteins ◽

Recent Work ◽

Integral Membrane Proteins ◽

Phospholipid Bilayer ◽

Endoplasmic Reticulum Membrane ◽

Regulatory Processes ◽

C Terminus ◽

Hydrophobic Amino Acids

A group of integral membrane proteins, known as C-tail anchored, is defined by the presence of a cytosolic N-terminal domain that is anchored to the phospholipid bilayer by a single segment of hydrophobic amino acids close to the C-terminus. The mode of insertion into membranes of these proteins, many of which play key roles in fundamental intracellular processes, is obligatorily post-translational, is highly specific and may be subject to regulatory processes that modulate the protein's function. Recent work has demonstrated that tail-anchored proteins translocate their C-termini across the endoplasmic reticulum membrane by a mechanism different from that used for Sec61-dependent post-translational signal-peptide-driven translocation. Here we summarize recent results on the insertion of tail-anchored proteins and discuss possible mechanisms that could be involved.

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Characterization of Constitutive macro-ER-phagy

10.1101/2020.10.21.349167 ◽

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.

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The yeast ERAD-C ubiquitin ligase Doa10 recognizes an intramembrane degron

The Journal of Cell Biology ◽

10.1083/jcb.201408088 ◽

2015 ◽

Vol 209 (2) ◽

pp. 261-273 ◽

Cited By ~ 36

Author(s):

Gregor Habeck ◽

Felix A. Ebner ◽

Hiroko Shimada-Kreft ◽

Stefan G. Kreft

Keyword(s):

Saccharomyces Cerevisiae ◽

Endoplasmic Reticulum ◽

Membrane Proteins ◽

Ubiquitin Ligase ◽

Protein Translocation ◽

Dependent Manner ◽

Β Subunit ◽

Human Orthologue ◽

Protein Ligases ◽

Prevailing View

Aberrant endoplasmic reticulum (ER) proteins are eliminated by ER-associated degradation (ERAD). This process involves protein retrotranslocation into the cytosol, ubiquitylation, and proteasomal degradation. ERAD substrates are classified into three categories based on the location of their degradation signal/degron: ERAD-L (lumen), ERAD-M (membrane), and ERAD-C (cytosol) substrates. In Saccharomyces cerevisiae, the membrane proteins Hrd1 and Doa10 are the predominant ERAD ubiquitin-protein ligases (E3s). The current notion is that ERAD-L and ERAD-M substrates are exclusively handled by Hrd1, whereas ERAD-C substrates are recognized by Doa10. In this paper, we identify the transmembrane (TM) protein Sec61 β-subunit homologue 2 (Sbh2) as a Doa10 substrate. Sbh2 is part of the trimeric Ssh1 complex involved in protein translocation. Unassembled Sbh2 is rapidly degraded in a Doa10-dependent manner. Intriguingly, the degron maps to the Sbh2 TM region. Thus, in contrast to the prevailing view, Doa10 (and presumably its human orthologue) has the capacity for recognizing intramembrane degrons, expanding its spectrum of substrates.

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Stitching proteins into membranes, not sew simple

Biological Chemistry ◽

10.1515/hsz-2014-0205 ◽

2014 ◽

Vol 395 (12) ◽

pp. 1417-1424 ◽

Cited By ~ 6

Author(s):

Paul Whitley ◽

Ismael Mingarro

Keyword(s):

Endoplasmic Reticulum ◽

Membrane Proteins ◽

Membrane Protein ◽

Protein Assembly ◽

Integral Membrane Proteins ◽

Eukaryotic Cells ◽

Endomembrane System ◽

Correct Orientation ◽

Tm Domain ◽

Er Membrane

Abstract Most integral membrane proteins located within the endomembrane system of eukaryotic cells are first assembled co-translationally into the endoplasmic reticulum (ER) before being sorted and trafficked to other organelles. The assembly of membrane proteins is mediated by the ER translocon, which allows passage of lumenal domains through and lateral integration of transmembrane (TM) domains into the ER membrane. It may be convenient to imagine multi-TM domain containing membrane proteins being assembled by inserting their first TM domain in the correct orientation, with subsequent TM domains inserting with alternating orientations. However a simple threading model of assembly, with sequential insertion of one TM domain into the membrane after another, does not universally stand up to scrutiny. In this article we review some of the literature illustrating the complexities of membrane protein assembly. We also present our own thoughts on aspects that we feel are poorly understood. In short we hope to convince the readers that threading of membrane proteins into membranes is ‘not sew simple’ and a topic that requires further investigation.

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Reduced temperature does not prevent transport of lysosomal integral membrane proteins from endoplasmic reticulum and through the Golgi system to lysosomes

European Journal of Biochemistry ◽

10.1111/j.1432-1033.1989.tb14942.x ◽

1989 ◽

Vol 183 (2) ◽

pp. 407-412 ◽

Cited By ~ 2

Author(s):

Pilar Garcia MORALES ◽

Javier G. BARRIOCANAL ◽

Ignacio V. SANDOVAL

Keyword(s):

Endoplasmic Reticulum ◽

Membrane Proteins ◽

Integral Membrane Proteins ◽

Golgi System ◽

Reduced Temperature

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An ATP-binding membrane protein is required for protein translocation across the endoplasmic reticulum membrane.

Cell Regulation ◽

10.1091/mbc.2.10.851 ◽

1991 ◽

Vol 2 (10) ◽

pp. 851-859 ◽

Cited By ~ 12

Author(s):

D L Zimmerman ◽

P Walter

Keyword(s):

Endoplasmic Reticulum ◽

Membrane Proteins ◽

Membrane Protein ◽

Protein Translocation ◽

Signal Sequence ◽

Microsomal Membrane ◽

Atp Binding ◽

Endoplasmic Reticulum Membrane ◽

Receptor Complex

The role of nucleotides in providing energy for polypeptide transfer across the endoplasmic reticulum (ER) membrane is still unknown. To address this question, we treated ER-derived mammalian microsomal vesicles with a photoactivatable analogue of ATP, 8-N3ATP. This treatment resulted in a progressive inhibition of translocation activity. Approximately 20 microsomal membrane proteins were labeled by [alpha 32P]8-N3ATP. Two of these were identified as proteins with putative roles in translocation, alpha signal sequence receptor (SSR), the 35-kDa subunit of the signal sequence receptor complex, and ER-p180, a putative ribosome receptor. We found that there was a positive correlation between inactivation of translocation activity and photolabeling of alpha SSR. In contrast, our data demonstrate that the ATP-binding domain of ER-p180 is dispensable for translocation activity and does not contribute to the observed 8-N3ATP sensitivity of the microsomal vesicles.

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Genes that control the fidelity of endoplasmic reticulum to Golgi transport identified as suppressors of vesicle budding mutations.

Molecular Biology of the Cell ◽

10.1091/mbc.7.7.1043 ◽

1996 ◽

Vol 7 (7) ◽

pp. 1043-1058 ◽

Cited By ~ 125

Author(s):

M J Elrod-Erickson ◽

C A Kaiser

Keyword(s):

Endoplasmic Reticulum ◽

Membrane Proteins ◽

Integral Membrane Proteins ◽

Secretory Proteins ◽

Vesicle Formation ◽

Gene Products ◽

Vesicle Budding ◽

Golgi Transport ◽

Transport Vesicles ◽

Copii Vesicle

Although convergent evidence suggests that proteins destined for export from the endoplasmic reticulum (ER) are separated from resident ER proteins and are concentrated into transport vesicles, the proteins that regulate this process have remained largely unknown. In a screen for suppressors of mutations in the essential COPII gene SEC13, we identified three genes (BST1, BST2/EMP24, and BST3) that negatively regulate COPII vesicle formation, preventing the production of vesicles with defective or missing subunits. Mutations in these genes slow the secretion of some secretory proteins and cause the resident ER proteins Kar2p and Pdi1p to leak more rapidly from the ER, indicating that these genes are also required for proper discrimination between resident ER proteins and Golgi-bound cargo molecules. The BST1 and BST2/EMP24 genes code for integral membrane proteins that reside predominantly in the ER. Our data suggest that the BST gene products represent a novel class of ER proteins that link the regulation of vesicle coat assembly to cargo sorting.

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