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

2013 ◽  
Vol 24 (19) ◽  
pp. 3123-3132 ◽  
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.

scholarly journals Two translocating hydrophilic segments of a nascent chain span the ER membrane during multispanning protein topogenesis

2007 ◽  
Vol 179 (7) ◽  
pp. 1441-1452 ◽  
Author(s):  
Yuichiro Kida ◽  
Fumiko Morimoto ◽  
Masao Sakaguchi
Keyword(s):  
Type I ◽  
Free System ◽  
Binding Peptide ◽  
Terminal Domain ◽  
N Terminus ◽  
Nascent Chain ◽  
A Cell ◽  
Signal Anchor ◽  
Anchor Sequence ◽  
Streptavidin Binding

During protein integration into the endoplasmic reticulum, the N-terminal domain preceding the type I signal-anchor sequence is translocated through a translocon. By fusing a streptavidin-binding peptide tag to the N terminus, we created integration intermediates of multispanning membrane proteins. In a cell-free system, N-terminal domain (N-domain) translocation was arrested by streptavidin and resumed by biotin. Even when N-domain translocation was arrested, the second hydrophobic segment mediated translocation of the downstream hydrophilic segment. In one of the defined intermediates, two hydrophilic segments and two hydrophobic segments formed a transmembrane disposition in a productive state. Both of the translocating hydrophilic segments were crosslinked with a translocon subunit, Sec61α. We conclude that two translocating hydrophilic segment in a single membrane protein can span the membrane during multispanning topogenesis flanking the translocon. Furthermore, even after six successive hydrophobic segments entered the translocon, N-domain translocation could be induced to restart from an arrested state. These observations indicate the remarkably flexible nature of the translocon.

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

2000 ◽  
Vol 150 (4) ◽  
pp. 719-730 ◽  
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.

scholarly journals Coupled Translocation Events Generate Topological Heterogeneity at the Endoplasmic Reticulum Membrane

1998 ◽  
Vol 9 (9) ◽  
pp. 2681-2697 ◽  
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.

scholarly journals Endoplasmic Reticulum Localization of Gaa1 and PIG-T, Subunits of the Glycosylphosphatidylinositol Transamidase Complex

2005 ◽  
Vol 280 (16) ◽  
pp. 16402-16409 ◽  
Author(s):  
Saulius Vainauskas ◽  
Anant K. Menon
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Fusion Proteins ◽  
Membrane Glycoprotein ◽  
Type I ◽  
N Terminus ◽  
Localization Signal ◽  
Tm Domain ◽  
Transmembrane Span ◽  
Er Membrane

After integration into the endoplasmic reticulum (ER) membrane, ER-resident membrane proteins must be segregated from proteins that are exported to post-ER compartments. Here we analyze how human Gaa1 and PIG-T, two of the five subunits of the ER-localized glycosylphosphatidylinositol transamidase complex, are retained in the ER. Neither protein contains a known ER localization signal. Gaa1 is a polytopic membrane glycoprotein with a cytoplasmic N terminus and a large luminal loop between its first two transmembrane spans; PIG-T is a type I membrane glycoprotein. To simplify our analyses, we studied Gaa1 and PIG-T constructs that could not interact with other subunits of the transamidase. We now show that Gaa1282, a truncated protein consisting of the first TM domain and luminal loop of Gaa1, is correctly oriented,N-glycosylated, and ER-localized. Removal of a potential ER localization signal in the form of a triple arginine cluster near the N terminus of Gaa1 or Gaa1282had no effect on ER localization. Fusion proteins consisting of different elements of Gaa1282appended to α2,6-sialyltransferase or transferrin receptor could exit the ER, indicating that Gaa1282, and by implication Gaa1, does not contain any dominant ER-sorting determinants. The data suggest that Gaa1 is passively retained in the ER by a signalless mechanism. In contrast, similar analyses of PIG-T revealed that it is ER-localized because of information in its transmembrane span; fusion of the PIG-T transmembrane span to Tac antigen, a plasma membrane-localized protein, caused the fusion protein to remain in the ER. These data are discussed in the context of models that have been proposed to account for retention of ER membrane proteins.

scholarly journals Stability and flexibility of marginally hydrophobic–segment stalling at the endoplasmic reticulum translocon

2016 ◽  
Vol 27 (6) ◽  
pp. 930-940 ◽  
Author(s):  
Yuichiro Kida ◽  
Yudai Ishihara ◽  
Hidenobu Fujita ◽  
Yukiko Onishi ◽  
Masao Sakaguchi
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Lipid Phase ◽  
Endoplasmic Reticulum Membrane ◽  
Dependent Manner ◽  
Transmembrane Segment ◽  
Conducting Channel ◽  
Transmembrane Segments ◽  
Lipid Environment ◽  
Sec61 Channel

Many membrane proteins are integrated into the endoplasmic reticulum membrane through the protein-conducting channel, the translocon. Transmembrane segments with insufficient hydrophobicity for membrane integration are frequently found in multispanning membrane proteins, and such marginally hydrophobic (mH) segments should be accommodated, at least transiently, at the membrane. Here we investigated how mH-segments stall at the membrane and their stability. Our findings show that mH-segments can be retained at the membrane without moving into the lipid phase and that such segments flank Sec61α, the core channel of the translocon, in the translational intermediate state. The mH-segments are gradually transferred from the Sec61 channel to the lipid environment in a hydrophobicity-dependent manner, and this lateral movement may be affected by the ribosome. In addition, stalling mH-segments allow for insertion of the following transmembrane segment, forming an Ncytosol/Clumen orientation, suggesting that mH-segments can move laterally to accommodate the next transmembrane segment. These findings suggest that mH-segments may be accommodated at the ER membrane with lateral fluctuation between the Sec61 channel and the lipid phase.

scholarly journals The catalytic activity of the endoplasmic reticulum-resident protein microsomal epoxide hydrolase towards carcinogens is retained on inversion of its membrane topology

1996 ◽  
Vol 319 (1) ◽  
pp. 131-136 ◽  
Author(s):  
Thomas FRIEDBERG ◽  
Romy HOLLER ◽  
Bettina LÖLLMANN ◽  
Michael ARAND ◽  
Franz OESCH
Keyword(s):  
Endoplasmic Reticulum ◽  
Catalytic Activity ◽  
Epoxide Hydrolase ◽  
Membrane Topology ◽  
Microsomal Epoxide Hydrolase ◽  
Type I ◽  
Type Ii ◽  
Membrane Orientation ◽  
N Terminus ◽  
Cytochrome P 450

Diol epoxides formed by the sequential action of cytochrome P-450 and the microsomal epoxide hydrolase (mEH) in the endoplasmic reticulum (ER) represent an important class of ultimate carcinogenic metabolites of polycyclic aromatic hydrocarbons. The role of the membrane orientation of cytochrome P-450 and mEH relative to each other in this catalytic cascade is not known. Cytochrome P-450 is known to have a type I topology. According to the algorithm of Hartman, Rapoport and Lodish [(1989) Proc. Natl. Acad. Sci. U.S.A. 86, 5786–5790], which allows the prediction of the membrane topology of proteins, mEH should adopt a type II membrane topology. Experimentally, mEH membrane topology has been disputed. Here we demonstrate that, in contrast with the theoretical prediction, the rat mEH has exclusively a type I membrane topology. Moreover we show that this topology can be inverted without affecting the catalytic activity of mEH. Our conclusions are supported by the observation that two mEH constructs (mEHg1 and mEHg2), containing engineered potential glycosylation sites at two separate locations after the C-terminal site of the membrane anchor, were not glycosylated in fibroblasts. However, changing the net charge at the N-terminus of these engineered mEH proteins by +3 resulted in proteins (++mEHg1 and ++mEHg2) that became glycosylated and consequently had a type II topology. The sensitivity of these glycosylated proteins to endoglycosidase H indicated that, like the native mEH, they are still retained in the ER. The engineered mEH proteins were integrated into membranes as they were resistant to alkaline extraction. Interestingly, an insect mEH with a charge distribution in its N-terminus similar to ++mEHg1 has recently been isolated. This enzyme might well display a type II topology instead of the type I topology of the rat mEH. Importantly, mEHg1, having the natural cytosolic orientation, as well as ++mEHg1, having an artificial luminal orientation, displayed rather similar substrate turnovers for the mutagenic metabolite benzo[a]pyrene 4,5-oxide. To our knowledge this is the first report demonstrating that topological inversion of a protein within the membrane of the ER has only a moderate effect on its enzymic activity, despite differences in folding pathways and redox environments on each side of the membrane. This observation represents an important step in the evaluation of the influence of mEH membrane orientation in the cascade of events leading to the formation of ultimate carcinogenic metabolites, and for studying the general importance of metabolic channelling on the surface of membranes.

scholarly journals Control of protein trafficking by reversible masking of transport signals

2016 ◽  
Vol 27 (8) ◽  
pp. 1310-1319 ◽  
Author(s):  
Omer Abraham ◽  
Karnit Gotliv ◽  
Anna Parnis ◽  
Gaelle Boncompain ◽  
Franck Perez ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Trafficking ◽  
Protein Transport ◽  
Protein Import ◽  
Binding Peptide ◽  
Protein Traffic ◽  
Alternative Approach ◽  
Targeting Signal ◽  
Regulated Trafficking ◽  
Streptavidin Binding

Systems that allow the control of protein traffic between subcellular compartments have been valuable in elucidating trafficking mechanisms. Most current approaches rely on ligand or light-controlled dimerization, which results in either retardation or enhancement of the transport of a reporter. We developed an alternative approach for trafficking regulation that we term “controlled unmasking of targeting elements” (CUTE). Regulated trafficking is achieved by reversible masking of the signal that directs the reporter to its target organelle, relying on the streptavidin–biotin system. The targeting signal is generated within or immediately after a 38–amino acid streptavidin-binding peptide (SBP) that is appended to the reporter. The binding of coexpressed streptavidin to SBP causes signal masking, whereas addition of biotin causes complex dissociation and triggers protein transport to the target organelle. We demonstrate the application of this approach to the control of nuclear and peroxisomal protein import and the generation of biotin-dependent trafficking through the endocytic and COPI systems. By simultaneous masking of COPI and endocytic signals, we were able to generate a synthetic pathway for efficient transport of a reporter from the plasma membrane to the endoplasmic reticulum.

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