scholarly journals Translocation of the C Terminus of a Tail-anchored Protein across the Endoplasmic Reticulum Membrane in Yeast Mutants Defective in Signal Peptide-driven Translocation

2002 ◽  
Vol 278 (5) ◽  
pp. 3489-3496 ◽  
Author(s):  
Monica Yabal ◽  
Silvia Brambillasca ◽  
Paolo Soffientini ◽  
Emanuela Pedrazzini ◽  
Nica Borgese ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Signal Peptide ◽  
Endoplasmic Reticulum Membrane ◽  
C Terminus ◽  
Yeast Mutants

Identification of topological determinants in the N-terminal domain of transcription factor Nrf1 that control its orientation in the endoplasmic reticulum membrane

2010 ◽  
Vol 430 (3) ◽  
pp. 497-510 ◽  
Author(s):  
Yiguo Zhang ◽  
John D. Hayes
Keyword(s):  
Amino Acids ◽  
Endoplasmic Reticulum ◽  
Transcription Factor ◽  
Signal Peptide ◽  
Leucine Zipper ◽  
Endoplasmic Reticulum Membrane ◽  
Related Factor ◽  
Terminal Domain ◽  
C Terminus ◽  
Er Membrane

Nrf1 [NF-E2 (nuclear factor-erythroid 2)-related factor 1] is a CNC (cap'n'collar) bZIP (basic-region leucine zipper) transcription factor that is tethered to ER (endoplasmic reticulum) and nuclear envelope membranes through its N-terminal signal peptide (residues 1–30). Besides the signal peptide, amino acids 31–90 of Nrf1 also negatively regulate the CNC-bZIP factor. In the present study we have tested the hypothesis that amino acids 31–90 of Nrf1, and the overlapping NHB2 (N-terminal homology box 2; residues 82–106), inhibit Nrf1 because they control its topology within membranes. This region contains three amphipathic α-helical regions comprising amino acids 31–50 [called the SAS (signal peptide-associated sequence)], 55–82 [called the CRACs (cholesterol-recognition amino acid consensus sequences)] and 89–106 (part of NHB2). We present experimental data showing that the signal peptide of Nrf1 contains a TM1 (transmembrane 1) region (residues 7–24) that is orientated across the ER membrane in an Ncyt/Clum fashion with its N-terminus facing the cytoplasm and its C-terminus positioned in the lumen of the ER. Once Nrf1 is anchored to the ER membrane through TM1, the remaining portion of the N-terminal domain (NTD, residues 1–124) is transiently translocated into the ER lumen. Thereafter, Nrf1 adopts a topology in which the SAS is inserted into the membrane, the CRACs are probably repartitioned to the cytoplasmic side of the ER membrane, and NHB2 may serve as an anchor switch, either lying on the luminal surface of the ER or traversing the membrane with an Ncyt/Clum orientation. Thus Nrf1 can adopt several topologies within membranes that are determined by its NTD.

Intramembrane proteolysis and post-targeting functions of signal peptides

2003 ◽  
Vol 31 (6) ◽  
pp. 1243-1247 ◽  
Author(s):  
B. Martoglio
Keyword(s):  
Endoplasmic Reticulum ◽  
Lipid Bilayer ◽  
Signal Peptide ◽  
Eukaryotic Cells ◽  
Endoplasmic Reticulum Membrane ◽  
Signal Peptides ◽  
Peptide Fragments ◽  
Intramembrane Proteolysis ◽  
Signal Sequences ◽  
Membrane Spanning

Signal sequences are the addresses of proteins destined for secretion. In eukaryotic cells, they mediate targeting to the endoplasmic reticulum membrane and insertion into the translocon. Thereafter, signal sequences are cleaved from the pre-protein and liberated into the endoplasmic reticulum membrane. We have recently reported that some liberated signal peptides are further processed by the intramembrane-cleaving aspartic protease signal peptide peptidase. Cleavage in the membrane-spanning portion of the signal peptide promotes the release of signal peptide fragments from the lipid bilayer. Typical processes that include intramembrane proteolysis is the regulatory or signalling function of cleavage products. Likewise, signal peptide fragments liberated upon intramembrane cleavage may promote such post-targeting functions in the cell.

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 TheSaccharomyces cerevisiaePrenylcysteine Carboxyl Methyltransferase Ste14p Is in the Endoplasmic Reticulum Membrane

1998 ◽  
Vol 9 (8) ◽  
pp. 2231-2247 ◽  
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.

scholarly journals NHE6 Protein Possesses a Signal Peptide Destined for Endoplasmic Reticulum Membrane and Localizes in Secretory Organelles of the Cell

2001 ◽  
Vol 276 (52) ◽  
pp. 49221-49227 ◽  
Author(s):  
Emi Miyazaki ◽  
Masao Sakaguchi ◽  
Shigeo Wakabayashi ◽  
Munekazu Shigekawa ◽  
Katsuyoshi Mihara
Keyword(s):  
Endoplasmic Reticulum ◽  
Signal Peptide ◽  
Endoplasmic Reticulum Membrane

Biogenesis of tail-anchored proteins

2003 ◽  
Vol 31 (6) ◽  
pp. 1238-1242 ◽  
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.

scholarly journals A Defective Signal Peptide Tethers the floury-2 Zein to the Endoplasmic Reticulum Membrane

1997 ◽  
Vol 114 (1) ◽  
pp. 345-352 ◽  
Author(s):  
J. W. Gillikin ◽  
F. Zhang ◽  
C. E. Coleman ◽  
H. W. Bass ◽  
B. A. Larkins ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Signal Peptide ◽  
Endoplasmic Reticulum Membrane
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