scholarly journals A nascent membrane protein is located adjacent to ER membrane proteins throughout its integration and translation.

1991 ◽  
Vol 112 (5) ◽  
pp. 809-821 ◽  
Author(s):  
R N Thrift ◽  
D W Andrews ◽  
P Walter ◽  
A E Johnson
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Transmembrane Domain ◽  
Signal Sequence ◽  
Secretory Protein ◽  
In Vitro Translation ◽  
Nascent Chain ◽  
Transmembrane Sequence ◽  
Er Membrane

The immediate environment of nascent membrane proteins undergoing integration into the ER membrane was investigated by photocrosslinking. Nascent polypeptides of different lengths, each containing a single IgM transmembrane sequence that functions either as a stop-transfer or a signal-anchor sequence, were synthesized by in vitro translation of truncated mRNAs in the presence of N epsilon-(5-azido-2-nitrobenzoyl)-Lys-tRNA, signal recognition particle, and microsomal membranes. This yielded nascent chains with photoreactive probes at one end of the transmembrane sequence where two lysine residues are located. When irradiated, these nascent chains reacted covalently with several ER proteins. One prominent crosslinking target was a glycoprotein similar in size to a protein termed mp39, shown previously to be situated adjacent to a secretory protein during its translocation across the ER membrane (Krieg, U. C., A. E. Johnson, and P. Walter. 1989. J. Cell Biol. 109:2033-2043; Wiedmann, M., D. Goerlich, E. Hartmann, T. V. Kurzchalia, and T. A. Rapoport. 1989. FEBS (Fed. Eur. Biochem. Soc.) Lett. 257:263-268) and likely to be identical to a protein previously designated the signal sequence receptor (Wiedmann, M., T. V. Kurzchalia, E. Hartmann, and T. A. Rapoport. 1987. Nature (Lond.). 328:830-833). Changing the orientation of the transmembrane domain in the bilayer, or making the transmembrane domain the first topogenic sequence in the nascent chain instead of the second, did not significantly alter the identities of the ER proteins that were the primary crosslinking targets. Furthermore, the nascent chains crosslinked to the mp39-like glycoprotein and other microsomal proteins even after the cytoplasmic tail of the nascent chain had been lengthened by nearly 100 amino acids beyond the stop-transfer sequence. Yet when the nascent chain was allowed to terminate normally, the major photocrosslinks were no longer observed, including in particular that to the mp39-like glycoprotein. These results show that the transmembrane segment of a nascent membrane protein is located adjacent to the mp39-like glycoprotein and other ER proteins during the integration process, and that at least a portion of the nascent chain remains in close proximity to these ER proteins until translation has been completed.

scholarly journals Membrane integration of Sec61α: a core component of the endoplasmic reticulum translocation complex

1998 ◽  
Vol 331 (1) ◽  
pp. 161-167 ◽  
Author(s):  
Bruce C. KNIGHT ◽  
Stephen HIGH
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Protein ◽  
Transmembrane Domain ◽  
Insertion Site ◽  
Secretory Protein ◽  
Cross Linking ◽  
Central Component ◽  
Membrane Insertion ◽  
Er Membrane

The Sec61 complex is a central component of the endoplasmic reticulum (ER) translocation site. The complex consists of three subunits: Sec61α, Sec61β and Sec61γ, at least two of which (α and β) are adjacent to nascent proteins during membrane insertion. Another component of the translocation machinery is the translocating chain-associating membrane (TRAM) protein, which is also adjacent to many nascent proteins during membrane insertion. Sec61α functions as the major component of a transmembrane channel formed by oligomers of the Sec61 complex. This channel is the site of secretory protein translocation and membrane protein integration at the ER membrane. Sec61α is a polytopic integral membrane protein, and we have studied its biosynthesis and membrane integration in vitro. Using a cross-linking approach to analyse the environment of a series of discrete Sec61α membrane-integration intermediates, we find: (i) newly synthesized Sec61α is adjacent to known components of the ER membrane-insertion site, namely Sec61α, Sec61β and TRAM, and thus the integration of Sec61α appears to require a pre-existing Sec61 complex; (ii) a site-specific cross-linking analysis indicates that the first transmembrane domain of Sec61α remains adjacent to protein components of the ER-insertion site (specifically TRAM and Sec61β) during the insertion of at least three subsequent transmembrane domains; and (iii) the membrane integration of Sec61α requires ER targeting by the signal-recognition particle.

scholarly journals Protein translocation across the endoplasmic reticulum membrane: identification by photocross-linking of a 39-kD integral membrane glycoprotein as part of a putative translocation tunnel.

1989 ◽  
Vol 109 (5) ◽  
pp. 2033-2043 ◽  
Author(s):  
U C Krieg ◽  
A E Johnson ◽  
P Walter
Keyword(s):  
Protein Translocation ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
In Vitro Translation ◽  
Secretory Proteins ◽  
Endoplasmic Reticulum Membrane ◽  
Membrane Glycoprotein ◽  
Messenger Rnas ◽  
Er Membrane

The molecular environment of secretory proteins during translocation across the ER membrane was examined by photocross-linking. Nascent preprolactin chains of various lengths, synthesized by in vitro translation of truncated messenger RNAs in the presence of N epsilon-(5-azido-2-nitrobenzoyl)-Lys-tRNA, signal recognition particle, and microsomal membranes, were used to position photoreactive probes at various locations within the membrane. Upon photolysis, each nascent chain species was cross-linked to an integral membrane glycoprotein with a deduced mass of 39 kD (mp39) via photoreactive lysines located in either the signal sequence or the mature prolactin sequence. Thus, different portions of the nascent preprolactin chain are in close proximity to the same membrane protein during the course of translocation, and mp39 therefore appears to be part of the translocon, the specific site of protein translocation across the ER membrane. The similarity of the molecular and cross-linking properties of mp39 and the glyco-protein previously identified as a signal sequence receptor (Wiedmann, M., T. V. Kurzchalia, E. Hartmann, and T. A. Rapoport. 1987. Nature [Lond.]. 328: 830-833) suggests that these two proteins may be identical. Our data indicate, however, that mp39 does not (or not only) function as a signal sequence receptor, but rather may be part of a putative translocation tunnel.

scholarly journals Characterization of a component of the yeast secretion machinery: identification of the SEC18 gene product.

1988 ◽  
Vol 8 (10) ◽  
pp. 4098-4109 ◽  
Author(s):  
K A Eakle ◽  
M Bernstein ◽  
S D Emr
Keyword(s):  
Golgi Complex ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Signal Sequence ◽  
Secretory Protein ◽  
Yeast Cells ◽  
In Vitro Translation ◽  
Cell Fractionation ◽  
Significant Similarity

SEC18 gene function is required for secretory protein transport between the endoplasmic reticulum (ER) and the Golgi complex. We cloned the SEC18 gene by complementation of the sec18-1 mutation. Gene disruption has shown that SEC18 is essential for yeast cell growth. Sequence analysis of the gene revealed a 2,271-base-pair open reading frame which could code for a protein of 83.9 kilodaltons. The predicted protein sequence showed no significant similarity to other known protein sequences. In vitro transcription and translation of SEC18 led to the synthesis of two proteins of approximately 84 and 82 kilodaltons. Antisera raised against a Sec18-beta-galactosidase fusion protein also detected two proteins (collectively referred to as Sec18p) in extracts of 35S-labeled yeast cells identical in size to those seen by in vitro translation. Mapping of the 5' end of the SEC18 mRNA revealed only one major start site for transcription, which indicates that the multiple forms of Sec18p do not arise from mRNAs with different 5' ends. Results of pulse-chase experiments indicated that the two forms of Sec18p are not the result of posttranslational processing. We suggest that translation initiating at different in-frame AUG start codons is likely to account for the presence of two forms of Sec18p. Hydrophobicity analysis indicated that the proteins were hydrophilic in nature and lacked any region that would be predicted to serve as a signal sequence or transmembrane anchor. Although potential sites for N-linked glycosylation were present in the Sec18p sequence, the sizes of the in vivo SEC18 gene products were unaffected by the drug tunicamycin, indicating that Sec18p does not enter the secretory pathway. These results suggest that Sec18p resides in the cell cytoplasm. While preliminary cell fractionation studies showed that Sec18p is not associated with the ER or Golgi complex, association with a 100,000 x g pellet fraction was observed. This suggests that Sec18p may bind transiently to small vesicles such as those presumed to participate in secretory protein transport between ER and the Golgi complex.

scholarly journals Targeting of NH2-terminal–processed Microsomal Protein to Mitochondria: A Novel Pathway for the Biogenesis of Hepatic Mitochondrial P450MT2

1997 ◽  
Vol 139 (3) ◽  
pp. 589-599 ◽  
Author(s):  
Sankar Addya ◽  
Hindupur K. Anandatheerthavarada ◽  
Gopa Biswas ◽  
Shripad V. Bhagwat ◽  
Jayati Mullick ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Signal Sequence ◽  
Limited Proteolysis ◽  
Membrane Insertion ◽  
Sequence Motif ◽  
Specific Marker ◽  
Mitochondrial Targeting ◽  
Targeting Signal ◽  
Er Membrane

Cytochrome P4501A1 is a hepatic, microsomal membrane–bound enzyme that is highly induced by various xenobiotic agents. Two NH2-terminal truncated forms of this P450, termed P450MT2a and MT2b, are also found localized in mitochondria from β-naphthoflavone–induced livers. In this paper, we demonstrate that P4501A1 has a chimeric NH2-terminal signal that facilitates the targeting of the protein to both the ER and mitochondria. The NH2-terminal 30–amino acid stretch of P4501A1 is thought to provide signals for ER membrane insertion and also stop transfer. The present study provides evidence that a sequence motif immediately COOH-terminal (residues 33–44) to the transmembrane domain functions as a mitochondrial targeting signal under both in vivo and in vitro conditions, and that the positively charged residues at positions 34 and 39 are critical for mitochondrial targeting. Results suggest that 25% of P4501A1 nascent chains, which escape ER membrane insertion, are processed by a liver cytosolic endoprotease. We postulate that the NH2-terminal proteolytic cleavage activates a cryptic mitochondrial targeting signal. Immunofluorescence microscopy showed that a portion of transiently expressed P4501A1 is colocalized with the mitochondrial-specific marker protein cytochrome oxidase subunit I. The mitochondrial-associated MT2a and MT2b are localized within the inner membrane compartment, as tested by resistance to limited proteolysis in both intact mitochondria and mitoplasts. Our results therefore describe a novel mechanism whereby proteins with chimeric signal sequence are targeted to the ER as well as to the mitochondria.

scholarly journals The signal sequence receptor, unlike the signal recognition particle receptor, is not essential for protein translocation

1992 ◽  
Vol 117 (1) ◽  
pp. 15-25 ◽  
Author(s):  
G Migliaccio ◽  
CV Nicchitta ◽  
G Blobel
Keyword(s):  
Membrane Protein ◽  
Protein Translocation ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Chain Complex ◽  
Secretory Protein ◽  
Signal Recognition ◽  
Ribosome Binding ◽  
Signal Recognition Particle Receptor ◽  
Nascent Chain

Detergent extracts of canine pancreas rough microsomal membranes were depleted of either the signal recognition particle receptor (SR), which mediates the signal recognition particle (SRP)-dependent targeting of the ribosome/nascent chain complex to the membrane, or the signal sequence receptor (SSR), which has been proposed to function as a membrane bound receptor for the newly targeted nascent chain and/or as a component of a multi-protein translocation complex responsible for transfer of the nascent chain across the membrane. Depletion of the two components was performed by chromatography of detergent extracts on immunoaffinity supports. Detergent extracts lacking either SR or SSR were reconstituted and assayed for activity with respect to SR dependent elongation arrest release, nascent chain targeting, ribosome binding, secretory precursor translocation, and membrane protein integration. Depletion of SR resulted in the loss of elongation arrest release activity, nascent chain targeting, secretory protein translocation, and membrane protein integration, although ribosome binding was unaffected. Full activity was restored by addition of immunoaffinity purified SR before reconstitution of the detergent extract. Surprisingly, depletion of SSR was without effect on any of the assayed activities, indicating that SSR is either not required for translocation or is one of a family of functionally redundant components.

scholarly journals Current strategies for protein production and purification enabling membrane protein structural biology

2016 ◽  
Vol 94 (6) ◽  
pp. 507-527 ◽  
Author(s):  
Aditya Pandey ◽  
Kyungsoo Shin ◽  
Robin E. Patterson ◽  
Xiang-Qin Liu ◽  
Jan K. Rainey
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Structural Biology ◽  
Protein Production ◽  
Protein Structures ◽  
Data Bank ◽  
In Vitro Translation ◽  
Preparation Methods ◽  
Natural Hosts

Membrane proteins are still heavily under-represented in the protein data bank (PDB), owing to multiple bottlenecks. The typical low abundance of membrane proteins in their natural hosts makes it necessary to overexpress these proteins either in heterologous systems or through in vitro translation/cell-free expression. Heterologous expression of proteins, in turn, leads to multiple obstacles, owing to the unpredictability of compatibility of the target protein for expression in a given host. The highly hydrophobic and (or) amphipathic nature of membrane proteins also leads to challenges in producing a homogeneous, stable, and pure sample for structural studies. Circumventing these hurdles has become possible through the introduction of novel protein production protocols; efficient protein isolation and sample preparation methods; and, improvement in hardware and software for structural characterization. Combined, these advances have made the past 10–15 years very exciting and eventful for the field of membrane protein structural biology, with an exponential growth in the number of solved membrane protein structures. In this review, we focus on both the advances and diversity of protein production and purification methods that have allowed this growth in structural knowledge of membrane proteins through X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and cryo-electron microscopy (cryo-EM).

scholarly journals Rer1p, a Retrieval Receptor for ER Membrane Proteins, Recognizes Transmembrane Domains in Multiple Modes

2003 ◽  
Vol 14 (9) ◽  
pp. 3605-3616 ◽  
Author(s):  
Ken Sato ◽  
Miyuki Sato ◽  
Akihiko Nakano
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Transmembrane Domain ◽  
Multivesicular Body ◽  
Spatial Location ◽  
Hydrophobic Core ◽  
Polar Residues ◽  
Localization Signal ◽  
Highly Hydrophobic ◽  
Er Membrane

The yeast Golgi membrane protein Rer1p is required for the retrieval of various endoplasmic reticulum (ER) membrane proteins such as Sec12p and Sec71p to the ER. We demonstrate here that the transmembrane domain (TMD) of Sec71p, a type-III membrane protein, contains an ER localization signal, which is required for physical recognition by Rer1p. The Sec71TMD-GFP fusion protein is efficiently retrieved to the ER by Rer1p. The structural feature of this TMD signal turns out to be the spatial location of polar residues flanking the highly hydrophobic core sequence but not the whole length of the TMD. On the Rer1p side, Tyr152 residue in the 4th TMD is important for the recognition of Sec12p but not Sec71p, suggesting that Rer1p interacts with its ligands at least in two modes. Sec71TMD-GFP expressed in the Δrer1 mutant cells is mislocalized from the ER to the lumen of vacuoles via the multivesicular body (MVB) sorting pathway. In this case, not only the presence of polar residues in the Sec71TMD but also the length of the TMD is critical for the MVB sorting. Thus, the Rer1p-dependent ER retrieval and the MVB sorting in late endosomes both watch polar residues in the TMD but in a different manner.

scholarly journals Formation of a functional ribosome-membrane junction during translocation requires the participation of a GTP-binding protein.

1986 ◽  
Vol 103 (6) ◽  
pp. 2253-2261 ◽  
Author(s):  
T Connolly ◽  
R Gilmore
Keyword(s):  
Signal Sequence ◽  
Binding Protein ◽  
Signal Recognition Particle ◽  
Secretory Protein ◽  
Signal Recognition ◽  
Gtp Binding Protein ◽  
Gtp Binding ◽  
Protease Digestion ◽  
Nascent Chain

The requirement for ribonucleotides and ribonucleotide hydrolysis was examined at several distinct points during translocation of a secretory protein across the endoplasmic reticulum. We monitored binding of in vitro-assembled polysomes to microsomal membranes after removal of ATP and GTP. Ribonucleotides were not required for the initial low salt-insensitive attachment of the ribosome to the membrane. However, without ribonucleotides the nascent secretory chains were sensitive to protease digestion and were readily extracted from the membrane with either EDTA or 0.5 M KOAc. In contrast, nascent chains resisted extraction with either EDTA or 0.5 M KOAc and were insensitive to protease digestion after addition of GTP or nonhydrolyzable GTP analogues. Translocation of the nascent secretory polypeptide was detected only when ribosome binding was conducted in the presence of GTP. Thus, translocation-competent binding of the ribosome to the membrane requires the participation of a novel GTP-binding protein in addition to the signal recognition particle and the signal recognition particle receptor. The second event we examined was translocation and processing of a truncated secretory polypeptide. Membrane-bound polysomes bearing an 86-residue nascent chain were generated by translation of a truncated preprolactin mRNA. Ribonucleotide-independent translocation of the polypeptide was detected by cleavage of the 30-residue signal sequence after puromycin termination. Nascent chain transport, per se, is apparently dependent upon neither ribonucleotide hydrolysis nor continued elongation of the polypeptide once a functional ribosome-membrane junction has been established.

scholarly journals SecY alterations that impair membrane protein folding and generate a membrane stress

2007 ◽  
Vol 176 (3) ◽  
pp. 307-317 ◽  
Author(s):  
Nobuyuki Shimohata ◽  
Shushi Nagamori ◽  
Yoshinori Akiyama ◽  
H. Ronald Kaback ◽  
Koreaki Ito
Keyword(s):  
Protein Folding ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Signal Recognition Particle ◽  
Membrane Vesicles ◽  
In Vitro Translation ◽  
Membrane Protein Folding ◽  
Protein Biogenesis ◽  
Simple Reduction

We report on a class of Escherichia coli SecY mutants that impair membrane protein folding. The mutants also up-regulate the Cpx/σE stress response pathways. Similar stress induction was also observed in response to a YidC defect in membrane protein biogenesis but not in response to the signal recognition particle–targeting defect or in response to a simple reduction in the abundance of the translocon. Together with the previous contention that the Cpx system senses a protein abnormality not only at periplasmic and outer membrane locations but also at the plasma membrane, abnormal states of membrane proteins are postulated to be generated in these secY mutants. In support of this notion, in vitro translation, membrane integration, and folding of LacY reveal that mutant membrane vesicles allow the insertion of LacY but not subsequent folding into a normal conformation recognizable by conformation-specific antibodies. The results demonstrate that normal SecY function is required for the folding of membrane proteins after their insertion into the translocon.

scholarly journals Characterization of a component of the yeast secretion machinery: identification of the SEC18 gene product

1988 ◽  
Vol 8 (10) ◽  
pp. 4098-4109
Author(s):  
K A Eakle ◽  
M Bernstein ◽  
S D Emr
Keyword(s):  
Golgi Complex ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Signal Sequence ◽  
Secretory Protein ◽  
Yeast Cells ◽  
In Vitro Translation ◽  
Cell Fractionation ◽  
Significant Similarity

SEC18 gene function is required for secretory protein transport between the endoplasmic reticulum (ER) and the Golgi complex. We cloned the SEC18 gene by complementation of the sec18-1 mutation. Gene disruption has shown that SEC18 is essential for yeast cell growth. Sequence analysis of the gene revealed a 2,271-base-pair open reading frame which could code for a protein of 83.9 kilodaltons. The predicted protein sequence showed no significant similarity to other known protein sequences. In vitro transcription and translation of SEC18 led to the synthesis of two proteins of approximately 84 and 82 kilodaltons. Antisera raised against a Sec18-beta-galactosidase fusion protein also detected two proteins (collectively referred to as Sec18p) in extracts of 35S-labeled yeast cells identical in size to those seen by in vitro translation. Mapping of the 5' end of the SEC18 mRNA revealed only one major start site for transcription, which indicates that the multiple forms of Sec18p do not arise from mRNAs with different 5' ends. Results of pulse-chase experiments indicated that the two forms of Sec18p are not the result of posttranslational processing. We suggest that translation initiating at different in-frame AUG start codons is likely to account for the presence of two forms of Sec18p. Hydrophobicity analysis indicated that the proteins were hydrophilic in nature and lacked any region that would be predicted to serve as a signal sequence or transmembrane anchor. Although potential sites for N-linked glycosylation were present in the Sec18p sequence, the sizes of the in vivo SEC18 gene products were unaffected by the drug tunicamycin, indicating that Sec18p does not enter the secretory pathway. These results suggest that Sec18p resides in the cell cytoplasm. While preliminary cell fractionation studies showed that Sec18p is not associated with the ER or Golgi complex, association with a 100,000 x g pellet fraction was observed. This suggests that Sec18p may bind transiently to small vesicles such as those presumed to participate in secretory protein transport between ER and the Golgi complex.

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