Sec62 Protein Mediates Membrane Insertion and Orientation of Moderately Hydrophobic Signal Anchor Proteins in the Endoplasmic Reticulum (ER)

We have identified membrane components which are adjacent to type I and type II signal-anchor proteins during their insertion into the membrane of the ER. Using two different cross-linking approaches a 37-38-kD nonglycosylated protein, previously identified as P37 (High, S., D. Görlich, M. Wiedmann, T. A. Rapoport, and B. Dobberstein. 1991. J. Cell Biol. 113:35-44), was found adjacent to all the membrane inserted nascent chains used in this study. On the basis of immunoprecipitation, this ER protein was shown to be identical to the recently identified mammalian Sec61 protein. Thus, Sec61p is the principal cross-linking partner of both type I and type II signal-anchor proteins during their membrane insertion (this work), and of secretory proteins during their translocation (Görlich, D., S. Prehn, E. Hartmann, K.-U. Kalies, and T. A. Rapoport. 1992. Cell. 71:489-503). We propose that membrane proteins of both orientations, and secretory proteins employ the same ER translocation sites, and that Sec61p is a core component of these sites.

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Multiple Determinants Direct the Orientation of Signal–Anchor Proteins: The Topogenic Role of the Hydrophobic Signal Domain

The Journal of Cell Biology ◽

10.1083/jcb.137.3.555 ◽

1997 ◽

Vol 137 (3) ◽

pp. 555-562 ◽

Cited By ~ 102

Author(s):

Johanna M. Wahlberg ◽

Martin Spiess

Keyword(s):

Transmembrane Domain ◽

Membrane Insertion ◽

Endoplasmic Reticulum Membrane ◽

Terminal Sequence ◽

Protein Orientation ◽

Signal Anchor ◽

Membrane Spanning ◽

Anchor Proteins

The orientation of signal–anchor proteins in the endoplasmic reticulum membrane is largely determined by the charged residues flanking the apolar, membrane-spanning domain and is influenced by the folding properties of the NH2-terminal sequence. However, these features are not generally sufficient to ensure a unique topology. The topogenic role of the hydrophobic signal domain was studied in vivo by expressing mutants of the asialoglycoprotein receptor subunit H1 in COS-7 cells. By replacing the 19-residue transmembrane segment of wild-type and mutant H1 by stretches of 7–25 leucine residues, we found that the length and hydrophobicity of the apolar sequence significantly affected protein orientation. Translocation of the NH2 terminus was favored by long, hydrophobic sequences and translocation of the COOH terminus by short ones. The topogenic contributions of the transmembrane domain, the flanking charges, and a hydrophilic NH2-terminal portion were additive. In combination these determinants were sufficient to achieve unique membrane insertion in either orientation.

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Environmental Transition of Signal-Anchor Sequences during Membrane Insertion via the Endoplasmic Reticulum Translocon

Molecular Biology of the Cell ◽

10.1091/mbc.e09-08-0738 ◽

2010 ◽

Vol 21 (3) ◽

pp. 418-429 ◽

Cited By ~ 11

Author(s):

Yuichiro Kida ◽

Chisato Kume ◽

Maki Hirano ◽

Masao Sakaguchi

Keyword(s):

Endoplasmic Reticulum ◽

Membrane Insertion ◽

Aqueous Environment ◽

Affinity Tag ◽

Transmembrane Segments ◽

Terminal Domain ◽

Signal Anchor ◽

Hydrophobic Portion ◽

Anchor Sequence ◽

Polypeptide Chains

In biogenesis of membrane proteins on the endoplasmic reticulum, a protein-conducting channel called the translocon functions in both the membrane translocation of lumenal domains and the integration of transmembrane segments. Here we analyzed the environments of polypeptide chains during the processes by water-dependent alkylation of N-ethylmaleimide at site-directed Cys residues. Using the technique, the region embedded in the hydrophobic portion of the membrane within a signal-anchor sequence and its shortening by insertion of a Pro residue could be detected. When translocation of the N-terminal domain of the signal-anchor was arrested by trapping an N-terminally fused affinity tag sequence, the signal-anchor was susceptible to alkylation, indicating that its migration into the hydrophobic environment was also arrested. Furthermore, when the tag sequence was separated from the signal-anchor by insertion of a hydrophilic sequence, the signal-anchor became inaccessible to alkylation even in the N-terminally trapped state. This suggests that membrane integration of the signal-anchor synchronizes with partial translocation of its N-terminal domain. Additionally, in an integration intermediate of a membrane protein, both of the two translocation-arrested hydrophilic chains were in an aqueous environment flanking the translocon, suggesting that the translocon provides the hydrophilic pathway capable of at least two translocating chains.

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Requirements for the membrane insertion of signal-anchor type proteins.

The Journal of Cell Biology ◽

10.1083/jcb.113.1.25 ◽

1991 ◽

Vol 113 (1) ◽

pp. 25-34 ◽

Cited By ~ 47

Author(s):

S High ◽

N Flint ◽

B Dobberstein

Keyword(s):

Signal Recognition Particle ◽

Membrane Insertion ◽

Type I ◽

Type Ii ◽

Nascent Chain ◽

Identical Manner ◽

Signal Anchor ◽

Protein Components ◽

Anchor Sequence ◽

Anchor Proteins

Proteins which are inserted and anchored in the membrane of the ER by an uncleaved signal-anchor sequence can assume two final orientations. Type I signal-anchor proteins translocate the NH2 terminus across the membrane while type II signal-anchor proteins translocate the COOH terminus. We investigated the requirements for cytosolic protein components and nucleotides for the membrane targeting and insertion of single-spanning type I signal-anchor proteins. Besides the ribosome, signal recognition particle (SRP), GTP, and rough microsomes (RMs) no other components were found to be required. The GTP analogue GMPPNP could substitute for GTP in supporting the membrane insertion of IMC-CAT. By using a photocrosslinking assay we show that for secreted, type I and type II signal-anchor proteins the presence of both GTP and RMs is required for the release of the nascent chain from the 54-kD subunit of SRP. For two of the proteins studied the release of the nascent chain from SRP54 was accompanied by a new interaction with components of the ER. We conclude that the GTP-dependent release of the nascent chain from SRP54 occurs in an identical manner for each of the proteins studied.

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Faculty Opinions recommendation of The ubiquitin-like protein Plic-1 enhances the membrane insertion of GABAA receptors by increasing their stability within the endoplasmic reticulum.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1116341.572358 ◽

2008 ◽

Author(s):

Eleanor Lederer

Keyword(s):

Endoplasmic Reticulum ◽

Gabaa Receptors ◽

Membrane Insertion

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Membrane Topogenesis of a Type I Signal-Anchor Protein, Mouse Synaptotagmin Ii, on the Endoplasmic Reticulum

The Journal of Cell Biology ◽

10.1083/jcb.150.4.719 ◽

2000 ◽

Vol 150 (4) ◽

pp. 719-730 ◽

Cited By ~ 39

Author(s):

Yuichiro Kida ◽

Masao Sakaguchi ◽

Mitsunori Fukuda ◽

Katsuhiko Mikoshiba ◽

Katsuyoshi Mihara

Keyword(s):

Endoplasmic Reticulum ◽

Endoplasmic Reticulum Membrane ◽

Type I ◽

Hydrophobic Region ◽

Anchor Protein ◽

Nascent Polypeptide ◽

Charged Residues ◽

Er Targeting ◽

Signal Anchor ◽

Positively Charged

Synaptotagmin II is a type I signal-anchor protein, in which the NH2-terminal domain of 60 residues (N-domain) is located within the lumenal space of the membrane and the following hydrophobic region (H-region) shows transmembrane topology. We explored the early steps of cotranslational integration of this molecule on the endoplasmic reticulum membrane and demonstrated the following: (a) The translocation of the N-domain occurs immediately after the H-region and the successive positively charged residues emerge from the ribosome. (b) Positively charged residues that follow the H-region are essential for maintaining the correct topology. (c) It is possible to dissect the lengths of the nascent polypeptide chains which are required for ER targeting of the ribosome and for translocation of the N-domain, thereby demonstrating that different nascent polypeptide chain lengths are required for membrane targeting and N-domain translocation. (d) The H-region is sufficiently long for membrane integration. (e) Proline residues preceding H-region are critical for N-domain translocation, but not for ER targeting. The proline can be replaced with amino acid with low helical propensity.

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Regulated Targeting of BAX to Mitochondria

The Journal of Cell Biology ◽

10.1083/jcb.143.1.207 ◽

1998 ◽

Vol 143 (1) ◽

pp. 207-215 ◽

Cited By ~ 447

Author(s):

Ing Swie Goping ◽

Atan Gross ◽

Josée N. Lavoie ◽

Mai Nguyen ◽

Ronald Jemmerson ◽

...

Keyword(s):

Mitochondrial Membrane ◽

Transmembrane Domain ◽

Apoptotic Cell ◽

Membrane Insertion ◽

Cell Extracts ◽

Proapoptotic Protein ◽

Signal Anchor ◽

Discrete Domains

The proapoptotic protein BAX contains a single predicted transmembrane domain at its COOH terminus. In unstimulated cells, BAX is located in the cytosol and in peripheral association with intracellular membranes including mitochondria, but inserts into mitochondrial membranes after a death signal. This failure to insert into mitochondrial membrane in the absence of a death signal correlates with repression of the transmembrane signal-anchor function of BAX by the NH2-terminal domain. Targeting can be instated by deleting the domain or by replacing the BAX transmembrane segment with that of BCL-2. In stimulated cells, the contribution of the NH2 terminus of BAX correlates with further exposure of this domain after membrane insertion of the protein. The peptidyl caspase inhibitor zVAD-fmk partly blocks the stimulated mitochondrial membrane insertion of BAX in vivo, which is consistent with the ability of apoptotic cell extracts to support mitochondrial targeting of BAX in vitro, dependent on activation of caspase(s). Taken together, our results suggest that regulated targeting of BAX to mitochondria in response to a death signal is mediated by discrete domains within the BAX polypeptide. The contribution of one or more caspases may reflect an initiation and/or amplification of this regulated targeting.

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Intracellular processing and transport of NH2-terminally truncated forms of a hemagglutinin-neuraminidase type II glycoprotein.

The Journal of Cell Biology ◽

10.1083/jcb.111.1.31 ◽

1990 ◽

Vol 111 (1) ◽

pp. 31-44 ◽

Cited By ~ 3

Author(s):

M K Spriggs ◽

P L Collins

Keyword(s):

Signal Sequence ◽

Active Form ◽

Cytoplasmic Domain ◽

Biologically Active ◽

Membrane Insertion ◽

Type I ◽

Type Ii ◽

Membrane Anchor ◽

Amino Terminal ◽

Signal Anchor

Six amino-terminal deletion mutants of the NH2-terminally anchored (type II orientation) hemagglutinin-neuraminidase (HN) protein of parainfluenza virus type 3 were expressed in tissue culture by recombinant SV-40 viruses. The mutations consisted of progressive deletions of the cytoplasmic domain and, in some cases, of the hydrophobic signal/anchor. Three activities were dissociated for the signal/anchor: membrane insertion, translocation, and anchoring/transport. HN protein lacking the entire cytoplasmic tail was inserted efficiently into the membrane of the endoplasmic reticulum but was translocated inefficiently into the lumen. However, the small amounts that were successfully translocated appeared to be processed subsequently in a manner indistinguishable from that of parental HN. Thus, the cytoplasmic domain was not required for maturation of this type II glycoprotein. Progressive deletions into the membrane anchor restored efficient translocation, indicating that the NH2-terminal 44 amino acids were fully dispensable for membrane insertion and translocation and that a 10-amino acid hydrophobic signal sequence was sufficient for both activities. These latter HN molecules appeared to be folded authentically as assayed by hemagglutination activity, reactivity with a conformation-specific antiserum, correct formation of intramolecular disulfide bonds, and homooligomerization. However, most (85-90%) of these molecules accumulated in the ER. This showed that folding and oligomerization into a biologically active form, which presumably represents a virion spike, occurs essentially to completion within that compartment but is not sufficient for efficient transport through the exocytotic pathway. Protein transport also appeared to depend on the structure of the membrane anchor. These latter mutants were not stably integrated in the membrane, and the small proportion (10-15%) that was processed through the exocytotic pathway was secreted. The maturation steps and some of the effects of mutations described here for a type II glycoprotein resemble previous observations for prototypic type I glycoproteins and are indicative of close similarities in these processes for proteins of both membrane orientations.

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