Signal Anchor Sequence Provides Motive Force for Polypeptide Chain Translocation through the Endoplasmic Reticulum Membrane

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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Coupled Translocation Events Generate Topological Heterogeneity at the Endoplasmic Reticulum Membrane

Molecular Biology of the Cell ◽

10.1091/mbc.9.9.2681 ◽

1998 ◽

Vol 9 (9) ◽

pp. 2681-2697 ◽

Cited By ~ 29

Author(s):

Kenneth Moss ◽

Andrew Helm ◽

Yun Lu ◽

Alvina Bragin ◽

William R. Skach

Keyword(s):

Endoplasmic Reticulum ◽

Structural Features ◽

Endoplasmic Reticulum Membrane ◽

Type I ◽

Type Ii ◽

Hydrophobic Residues ◽

P Glycoprotein ◽

C Terminus ◽

Signal Anchor ◽

Hydrophilic Residues

Topogenic determinants that direct protein topology at the endoplasmic reticulum membrane usually function with high fidelity to establish a uniform topological orientation for any given polypeptide. Here we show, however, that through the coupling of sequential translocation events, native topogenic determinants are capable of generating two alternate transmembrane structures at the endoplasmic reticulum membrane. Using defined chimeric and epitope-tagged full-length proteins, we found that topogenic activities of two C-trans (type II) signal anchor sequences, encoded within the seventh and eighth transmembrane (TM) segments of human P-glycoprotein were directly coupled by an inefficient stop transfer (ST) sequence (TM7b) contained within the C-terminus half of TM7. Remarkably, these activities enabled TM7 to achieve both a single- and a double-spanning TM topology with nearly equal efficiency. In addition, ST and C-trans signal anchor activities encoded by TM8 were tightly linked to the weak ST activity, and hence topological fate, of TM7b. This interaction enabled TM8 to span the membrane in either a type I or a type II orientation. Pleiotropic structural features contributing to this unusual topogenic behavior included 1) a short, flexible peptide loop connecting TM7a and TM7b, 2) hydrophobic residues within TM7b, and 3) hydrophilic residues between TM7b and TM8.

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Intramembrane Proteolysis and Endoplasmic Reticulum Retention of Hepatitis C Virus Core Protein

Journal of Virology ◽

10.1128/jvi.78.12.6370-6380.2004 ◽

2004 ◽

Vol 78 (12) ◽

pp. 6370-6380 ◽

Cited By ~ 69

Author(s):

Kiyoko Okamoto ◽

Kohji Moriishi ◽

Tatsuo Miyamura ◽

Yoshiharu Matsuura

Keyword(s):

Endoplasmic Reticulum ◽

Hepatitis C ◽

Core Protein ◽

Signal Sequence ◽

Hydrophobic Region ◽

E1 Protein ◽

Hcv Core Protein ◽

Er Retention ◽

Signal Anchor ◽

Anchor Sequence

ABSTRACT Hepatitis C virus (HCV) core protein is suggested to localize to the endoplasmic reticulum (ER) through a C-terminal hydrophobic region that acts as a membrane anchor for core protein and as a signal sequence for E1 protein. The signal sequence of core protein is further processed by signal peptide peptidase (SPP). We examined the regions of core protein responsible for ER retention and processing by SPP. Analysis of the intracellular localization of deletion mutants of HCV core protein revealed that not only the C-terminal signal-anchor sequence but also an upstream hydrophobic region from amino acid 128 to 151 is required for ER retention of core protein. Precise mutation analyses indicated that replacement of Leu139, Val140, and Leu144 of core protein by Ala inhibited processing by SPP, but cleavage at the core-E1 junction by signal peptidase was maintained. Additionally, the processed E1 protein was translocated into the ER and glycosylated with high-mannose oligosaccharides. Core protein derived from the mutants was translocated into the nucleus in spite of the presence of the unprocessed C-terminal signal-anchor sequence. Although the direct association of core protein with a wild-type SPP was not observed, expression of a loss-of-function SPP mutant inhibited cleavage of the signal sequence by SPP and coimmunoprecipitation with unprocessed core protein. These results indicate that Leu139, Val140, and Leu144 in core protein play crucial roles in the ER retention and SPP cleavage of HCV core protein.

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Interactions between Sec Complex and Prepro-α-Factor during Posttranslational Protein Transport into the Endoplasmic Reticulum

Molecular Biology of the Cell ◽

10.1091/mbc.e03-06-0390 ◽

2004 ◽

Vol 15 (1) ◽

pp. 1-10 ◽

Cited By ~ 37

Author(s):

Kathrin Plath ◽

Barrie M. Wilkinson ◽

Colin J. Stirling ◽

Tom A. Rapoport

Keyword(s):

Endoplasmic Reticulum ◽

Protein Transport ◽

Polypeptide Chain ◽

Signal Sequence ◽

Cross Linking ◽

Endoplasmic Reticulum Membrane ◽

Transmembrane Segments ◽

Sequence Recognition ◽

Multiple Regions ◽

A Site

Posttranslational translocation of prepro-α-factor (ppαF) across the yeast endoplasmic reticulum membrane begins with the binding of the signal sequence to the Sec complex, a membrane component consisting of the trimeric Sec61p complex and the tetrameric Sec62p/63p complex. We show by photo-cross-linking that the signal sequence is bound directly to a site where it contacts simultaneously Sec61p and Sec62p, suggesting that there is a single signal sequence recognition step. We found no evidence for the simultaneous contact of the signal sequence with two Sec61p molecules. To identify transmembrane segments of Sec61p that line the actual translocation pore, a late translocation intermediate of ppαF was generated with photoreactive probes incorporated into the mature portion of the polypeptide. Cross-linking to multiple regions of Sec61p was observed. In contrast to the signal sequence, neighboring positions of the mature portion of ppαF had similar interactions with Sec61p. These data suggest that the channel pore is lined by several transmembrane segments, which have no significant affinity for the translocating polypeptide chain.

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Membrane translocation of lumenal domains of membrane proteins powered by downstream transmembrane sequences

Molecular Biology of the Cell ◽

10.1091/mbc.e13-04-0210 ◽

2013 ◽

Vol 24 (19) ◽

pp. 3123-3132 ◽

Cited By ~ 3

Author(s):

Takaaki Yabuki ◽

Fumiko Morimoto ◽

Yuichiro Kida ◽

Masao Sakaguchi

Keyword(s):

Endoplasmic Reticulum ◽

Membrane Proteins ◽

Endoplasmic Reticulum Membrane ◽

Type I ◽

Surface Plasmon Resonance Analysis ◽

Binding Peptide ◽

Resonance Analysis ◽

N Terminus ◽

Signal Anchor ◽

Streptavidin Binding

Translocation of the N-terminus of a type I signal anchor (SA-I) sequence across the endoplasmic reticulum membrane can be arrested by tagging with a streptavidin-binding peptide tag (SBP tag) and trapping by streptavidin. In the present study, we first examine the affinity required for the translocation arrest. When the SBP tag is serially truncated, the ability for arrest gradually decreases. Surface plasmon resonance analysis shows that an interaction as strong as 10−8 M or a smaller dissociation constant is required for trapping the topogenesis of a natural SA-I sequence. Such truncated tags, however, become effective by mutating the SA-I sequence, suggesting that the translocation motivation is considerably influenced by the properties of the SA-I sequence. In addition, we introduce the SBP tag into lumenal loops of a multispanning membrane protein, human erythrocyte band 3. Among the tagged loops between transmembrane 1 (TM1) and TM8, three loops are trapped by cytosolic streptavidin. These loops are followed by TM sequences possessing topogenic properties, like the SA-I sequence, and translocation of one loop is diminished by insertion of a proline into the following TM sequence. These findings suggest that the translocation of lumenal loops by SA-I–like TM sequences has a crucial role in topogenesis of multispanning membrane proteins.

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An Artificial Mitochondrial Tail Signal/Anchor Sequence Confirms a Requirement for Moderate Hydrophobicity for Targeting

Bioscience Reports ◽

10.1007/s10540-007-9061-0 ◽

2007 ◽

Vol 27 (6) ◽

pp. 385-401 ◽

Cited By ~ 17

Author(s):

Binks W. Wattenberg ◽

Denise Clark ◽

Stephanie Brock

Keyword(s):

Amino Acids ◽

Endoplasmic Reticulum ◽

Outer Membrane ◽

Helical Structure ◽

Mitochondrial Outer Membrane ◽

Carboxy Terminus ◽

Charged Amino Acids ◽

Hydrophobic Amino Acids ◽

Signal Anchor ◽

Anchor Sequence

Tail-anchored proteins are a group of membrane proteins oriented with their amino terminus in the cytoplasm and their carboxy terminus embedded in intracellular membranes. This group includes the apoptosis-mediating proteins of the Bcl-2 family as well as the vesicle targeting proteins of the SNARE group, among others. A stretch of hydrophobic amino acids at the extreme carboxy terminus of these proteins serves both as a membrane anchor and as a targeting signal. Tail-anchored proteins are differentially targeted to either the endoplasmic reticulum or the mitochondrial outer membrane and the mechanism which accomplishes this selective targeting is poorly understood. Here we define important characteristics of the signal/anchor region which directs proteins to the mitochondrial outer membrane. We have created an artificial sequence consisting of a stretch of 16 leucines bounded by positively charged amino acids. Using this template we demonstrate that moderate hydrophobicity distinguishes the mitochondrial tail-anchor sequence from that of the endoplasmic reticulum tail-anchor sequence. A change as small as introduction of a single polar residue into a sequence that otherwise targets to the endoplasmic reticulum can substantially switch targeting to the mitochondrial outer membrane. Further we show that a mitochondrially targeted tail-anchor has a higher propensity for the formation of alpha-helical structure than a sequence directing tail-anchored proteins to the endoplasmic reticulum.

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Membrane insertion of uracil permease, a polytopic yeast plasma membrane protein.

Molecular and Cellular Biology ◽

10.1128/mcb.11.2.1114 ◽

1991 ◽

Vol 11 (2) ◽

pp. 1114-1124 ◽

Cited By ~ 69

Author(s):

S Silve ◽

C Volland ◽

C Garnier ◽

R Jund ◽

M R Chevallier ◽

...

Keyword(s):

Plasma Membrane ◽

Terminal Segment ◽

Proteolytic Processing ◽

Endoplasmic Reticulum Membrane ◽

Transmembrane Segments ◽

C Terminus ◽

Signal Anchor ◽

Anchor Sequence

Uracil permease is a multispanning protein of the Saccharomyces cerevisiae plasma membrane which is encoded by the FUR4 gene and produced in limited amounts. It has a long N-terminal hydrophilic segment, which is followed by 10 to 12 putative transmembrane segments, and a hydrophilic C terminus. The protein carries seven potential N-linked glycosylation sites, three of which are in its N-terminal segment. Overexpression of this permease and specific antibodies were used to show that uracil permease undergoes neither N-linked glycosylation nor proteolytic processing. Uracil permease N-terminal segments of increasing lengths were fused to a reporter glycoprotein, acid phosphatase. The in vitro and in vivo fates of the resulting hybrid proteins were analyzed to identify the first signal anchor sequence of the permease and demonstrate the cytosolic orientation of its N-terminal hydrophilic sequence. In vivo insertion of the hybrid protein bearing the first signal anchor sequence of uracil permease into the endoplasmic reticulum membrane was severely blocked in sec61 and sec62 translocation mutants.

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A Few Positively Charged Residues Slow Movement of a Polypeptide Chain across the Endoplasmic Reticulum Membrane

Biochemistry ◽

10.1021/bi500649y ◽

2014 ◽

Vol 53 (33) ◽

pp. 5375-5383 ◽

Cited By ~ 4

Author(s):

Marifu Yamagishi ◽

Yukiko Onishi ◽

Shotaro Yoshimura ◽

Hidenobu Fujita ◽

Kenta Imai ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Polypeptide Chain ◽

Endoplasmic Reticulum Membrane ◽

Slow Movement ◽

Charged Residues ◽

Positively Charged

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Membrane insertion of uracil permease, a polytopic yeast plasma membrane protein

Molecular and Cellular Biology ◽

10.1128/mcb.11.2.1114-1124.1991 ◽

1991 ◽

Vol 11 (2) ◽

pp. 1114-1124

Author(s):

S Silve ◽

C Volland ◽

C Garnier ◽

R Jund ◽

M R Chevallier ◽

...

Keyword(s):

Plasma Membrane ◽

Terminal Segment ◽

Proteolytic Processing ◽

Endoplasmic Reticulum Membrane ◽

Transmembrane Segments ◽

C Terminus ◽

Signal Anchor ◽

Anchor Sequence

Uracil permease is a multispanning protein of the Saccharomyces cerevisiae plasma membrane which is encoded by the FUR4 gene and produced in limited amounts. It has a long N-terminal hydrophilic segment, which is followed by 10 to 12 putative transmembrane segments, and a hydrophilic C terminus. The protein carries seven potential N-linked glycosylation sites, three of which are in its N-terminal segment. Overexpression of this permease and specific antibodies were used to show that uracil permease undergoes neither N-linked glycosylation nor proteolytic processing. Uracil permease N-terminal segments of increasing lengths were fused to a reporter glycoprotein, acid phosphatase. The in vitro and in vivo fates of the resulting hybrid proteins were analyzed to identify the first signal anchor sequence of the permease and demonstrate the cytosolic orientation of its N-terminal hydrophilic sequence. In vivo insertion of the hybrid protein bearing the first signal anchor sequence of uracil permease into the endoplasmic reticulum membrane was severely blocked in sec61 and sec62 translocation mutants.

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