Protein translocation mutants defective in the insertion of integral membrane proteins into the endoplasmic reticulum.

Yeast mutants defective in the translocation of soluble secretory proteins into the lumen of the endoplasmic reticulum (sec61, sec62, sec63) are not impaired in the assembly and glycosylation of the type II membrane protein dipeptidylaminopeptidase B (DPAPB) or of a chimeric membrane protein consisting of the multiple membrane-spanning domain of yeast hydroxymethylglutaryl CoA reductase (HMG1) fused to yeast histidinol dehydrogenase (HIS4C). This chimera is assembled in wild-type or mutant cells such that the His4c protein is oriented to the ER lumen and thus is not available for conversion of cytosolic histidinol to histidine. Cells harboring the chimera have been used to select new translocation defective sec mutants. Temperature-sensitive lethal mutations defining two complementation groups have been isolated: a new allele of sec61 and a single isolate of a new gene sec65. The new isolates are defective in the assembly of DPAPB, as well as the secretory protein alpha-factor precursor. Thus, the chimeric membrane protein allows the selection of more restrictive sec mutations rather than defining genes that are required only for membrane protein assembly. The SEC61 gene was cloned, sequenced, and used to raise polyclonal antiserum that detected the Sec61 protein. The gene encodes a 53-kDa protein with five to eight potential membrane-spanning domains, and Sec61p antiserum detects an integral protein localized to the endoplasmic reticulum membrane. Sec61p appears to play a crucial role in the insertion of secretory and membrane polypeptides into the endoplasmic reticulum.

Download Full-text

Topology and functional domains of Sec63p, an endoplasmic reticulum membrane protein required for secretory protein translocation.

Molecular and Cellular Biology ◽

10.1128/mcb.12.7.3288 ◽

1992 ◽

Vol 12 (7) ◽

pp. 3288-3296 ◽

Cited By ~ 161

Author(s):

D Feldheim ◽

J Rothblatt ◽

R Schekman

Keyword(s):

Endoplasmic Reticulum ◽

Membrane Protein ◽

Protein Translocation ◽

Protein A ◽

Integral Membrane Protein ◽

Secretory Protein ◽

Carboxyl Terminus ◽

Endoplasmic Reticulum Membrane ◽

Cell Fractionation ◽

Network Cell

SEC63 encodes a protein required for secretory protein translocation into the endoplasmic reticulum (ER) of Saccharomyces cerevisiae (J. A. Rothblatt, R. J. Deshaies, S. L. Sanders, G. Daum, and R. Schekman, J. Cell Biol. 109:2641-2652, 1989). Antibody directed against a recombinant form of the protein detects a 73-kDa polypeptide which, by immunofluorescence microscopy, is localized to the nuclear envelope-ER network. Cell fractionation and protease protection experiments confirm the prediction that Sec63p is an integral membrane protein. A series of SEC63-SUC2 fusion genes was created to assess the topology of Sec63p within the ER membrane. The largest hybrid proteins are unglycosylated, suggesting that the carboxyl terminus of Sec63p faces the cytosol. Invertase fusion to a loop in Sec63p that is flanked by two putative transmembrane domains produces an extensively glycosylated hybrid protein. This loop, which is homologous to the amino terminus of the Escherichia coli heat shock protein, DnaJ, is likely to face the ER lumen. By analogy to the interaction of the DnaJ and Hsp70-like DnaK proteins in E. coli, the DnaJ loop of Sec63p may recruit luminal Hsp70 (BiP/GRP78/Kar2p) to the translocation apparatus. Mutations in two highly conserved positions of the DnaJ loop and short deletions of the carboxyl terminus inactivate Sec63p activity. Sec63p associates with several other proteins, including Sec61p, a 31.5-kDa glycoprotein, and a 23-kDa protein, and together with these proteins may constitute part of the polypeptide translocation apparatus. A nonfunctional DnaJ domain mutant allele does not interfere with the formation of the Sec63p/Sec61p/gp31.5/p23 complex.

Download Full-text

Topology and functional domains of Sec63p, an endoplasmic reticulum membrane protein required for secretory protein translocation

Molecular and Cellular Biology ◽

10.1128/mcb.12.7.3288-3296.1992 ◽

1992 ◽

Vol 12 (7) ◽

pp. 3288-3296

Author(s):

D Feldheim ◽

J Rothblatt ◽

R Schekman

Keyword(s):

Endoplasmic Reticulum ◽

Membrane Protein ◽

Protein Translocation ◽

Protein A ◽

Integral Membrane Protein ◽

Secretory Protein ◽

Carboxyl Terminus ◽

Endoplasmic Reticulum Membrane ◽

Cell Fractionation ◽

Network Cell

Download Full-text

Characterization of secretory protein translocation: ribosome-membrane interaction in endoplasmic reticulum.

The Journal of Cell Biology ◽

10.1083/jcb.103.1.241 ◽

1986 ◽

Vol 103 (1) ◽

pp. 241-253 ◽

Cited By ~ 73

Author(s):

M Hortsch ◽

D Avossa ◽

D I Meyer

Keyword(s):

Endoplasmic Reticulum ◽

Protein Translocation ◽

Signal Recognition Particle ◽

Secretory Protein ◽

Signal Peptidase ◽

Secretory Proteins ◽

Peptidase Activity ◽

Ribosome Binding ◽

Functional Inactivation ◽

Docking Protein

Secretory proteins are synthesized on ribosomes bound to the membrane of the endoplasmic reticulum (ER). After the selection of polysomes synthesizing secretory proteins and their direction to the membrane of the ER via signal recognition particle (SRP) and docking protein respectively, the polysomes become bound to the ER membrane via an unknown, protein-mediated mechanism. To identify proteins involved in protein translocation, beyond the (SRP-docking protein-mediated) recognition step, controlled proteolysis was used to functionally inactivate rough microsomes that had previously been depleted of docking protein. As the membranes were treated with increasing levels of protease, they lost their ability to be functionally reconstituted with the active cytoplasmic fragment of docking protein (DPf). This functional inactivation did not correlate with a loss of either signal peptidase activity, nor with the ability of the DPf to reassociate with the membrane. It did correlate, however, with a loss of the ability of the microsomes to bind ribosomes. Ribophorins are putative ribosome-binding proteins. Immunoblots developed with monoclonal antibodies against canine ribophorins I and II demonstrated that no correlation exists between the protease-induced inability to bind ribosomes and the integrity of the ribophorins. Ribophorin I was 85% resistant and ribophorin II 100% resistant to the levels of protease needed to totally eliminate ribosome binding. Moreover, no direct association was found between ribophorins and ribosomes; upon detergent solubilization at low salt concentrations, ribophorins could be sedimented in the presence or absence of ribosomes. Finally, the alkylating agent N-ethylmaleimide was shown to be capable of inhibiting translocation (beyond the SRP-docking protein-mediated recognition step), but had no affect on the ability of ribosomes to bind to ER membranes. We conclude that potentially two additional proteinaceous components, as yet unidentified, are involved in protein translocation. One is protease sensitive and possibly involved in ribosome binding, the other is N-ethylmaleimide sensitive and of unknown function.

Download Full-text

An ATP-binding membrane protein is required for protein translocation across the endoplasmic reticulum membrane.

Cell Regulation ◽

10.1091/mbc.2.10.851 ◽

1991 ◽

Vol 2 (10) ◽

pp. 851-859 ◽

Cited By ~ 12

Author(s):

D L Zimmerman ◽

P Walter

Keyword(s):

Endoplasmic Reticulum ◽

Membrane Proteins ◽

Membrane Protein ◽

Protein Translocation ◽

Signal Sequence ◽

Microsomal Membrane ◽

Atp Binding ◽

Endoplasmic Reticulum Membrane ◽

Receptor Complex

The role of nucleotides in providing energy for polypeptide transfer across the endoplasmic reticulum (ER) membrane is still unknown. To address this question, we treated ER-derived mammalian microsomal vesicles with a photoactivatable analogue of ATP, 8-N3ATP. This treatment resulted in a progressive inhibition of translocation activity. Approximately 20 microsomal membrane proteins were labeled by [alpha 32P]8-N3ATP. Two of these were identified as proteins with putative roles in translocation, alpha signal sequence receptor (SSR), the 35-kDa subunit of the signal sequence receptor complex, and ER-p180, a putative ribosome receptor. We found that there was a positive correlation between inactivation of translocation activity and photolabeling of alpha SSR. In contrast, our data demonstrate that the ATP-binding domain of ER-p180 is dispensable for translocation activity and does not contribute to the observed 8-N3ATP sensitivity of the microsomal vesicles.

Download Full-text

Visualization of Distinct Entities of the SecYEG Translocon during Translocation and Integration of Bacterial Proteins

Molecular Biology of the Cell ◽

10.1091/mbc.e08-08-0886 ◽

2009 ◽

Vol 20 (6) ◽

pp. 1804-1815 ◽

Cited By ~ 30

Author(s):

Diana Boy ◽

Hans-Georg Koch

Keyword(s):

Membrane Protein ◽

Secretory Protein ◽

Secretory Proteins ◽

Lipid Phase ◽

Endoplasmic Reticulum Membrane ◽

Major Protein ◽

Native Page ◽

Substrate Complex ◽

The Stability ◽

Polytopic Membrane Protein

The universally conserved SecYEG/Sec61 translocon constitutes the major protein-conducting channel in the cytoplasmic membrane of bacteria and the endoplasmic reticulum membrane of eukaryotes. It is engaged in both translocating secretory proteins across the membrane as well as in integrating membrane proteins into the lipid phase of the membrane. In the current study we have detected distinct SecYEG translocon complexes in native Escherichia coli membranes. Blue-Native-PAGE revealed the presence of a 200-kDa SecYEG complex in resting membranes. When the SecA-dependent secretory protein pOmpA was trapped inside the SecYEG channel, a smaller SecY-containing complex of ∼140-kDa was observed, which probably corresponds to a monomeric SecYEG–substrate complex. Trapping the SRP-dependent polytopic membrane protein mannitol permease in the SecYEG translocon, resulted in two complexes of 250 and 600 kDa, each containing both SecY and the translocon-associated membrane protein YidC. The appearance of both complexes was correlated with the number of transmembrane domains that were exposed during targeting of mannitol permease to the membrane. These results suggest that the assembly or the stability of the bacterial SecYEG translocon is influenced by the substrate that needs to be transported.

Download Full-text

Elongation arrest is not a prerequisite for secretory protein translocation across the microsomal membrane.

The Journal of Cell Biology ◽

10.1083/jcb.100.6.1913 ◽

1985 ◽

Vol 100 (6) ◽

pp. 1913-1921 ◽

Cited By ~ 88

Author(s):

V Siegel ◽

P Walter

Keyword(s):

Endoplasmic Reticulum ◽

Protein Translocation ◽

Signal Recognition Particle ◽

Integral Membrane Protein ◽

Secretory Protein ◽

Secretory Proteins ◽

Signal Recognition ◽

7Sl Rna ◽

Nascent Chain ◽

Specific Proteins

Signal recognition particle (SRP) is a ribonucleoprotein consisting of six distinct polypeptides and one molecule of small cytoplasmic 7SL RNA. It was previously shown to promote the co-translational translocation of secretory proteins across the endoplasmic reticulum by (a) arresting the elongation of the presecretory nascent chain at a specific point, and (b) interacting with the SRP receptor, an integral membrane protein of the endoplasmic reticulum which is active in releasing the elongation arrest. Recently a procedure was designed by which the particle could be disassembled into its protein and RNA components. We have further separated the SRP proteins into four homogeneous fractions. When recombined with each other and with 7SL RNA, they formed fully active SRP. Particles missing specific proteins were assembled in the hope that some of these would retain some functional activity. SRP(-9/14), the particle lacking the 9-kD and 14-kD polypeptides, was fully active in promoting translocation, but was completely inactive in elongation arrest. This implied that elongation arrest is not a prerequisite for protein translocation. SRP receptor was required for SRP(-9/14)-mediated translocation to occur, and thus must play some role in the translocation process in addition to releasing the elongation arrest.

Download Full-text

Role of 26S proteasome and HRD genes in the degradation of 3-hydroxy-3-methylglutaryl-CoA reductase, an integral endoplasmic reticulum membrane protein.

Molecular Biology of the Cell ◽

10.1091/mbc.7.12.2029 ◽

1996 ◽

Vol 7 (12) ◽

pp. 2029-2044 ◽

Cited By ~ 387

Author(s):

R Y Hampton ◽

R G Gardner ◽

J Rine

Keyword(s):

Endoplasmic Reticulum ◽

Membrane Protein ◽

Mevalonate Pathway ◽

26S Proteasome ◽

Negative Regulator ◽

Sterol Synthesis ◽

Endoplasmic Reticulum Membrane ◽

Coding Region ◽

Coa Reductase

3-hydroxy-3-methylglutaryl-CoA reductase (HMG-R), a key enzyme of sterol synthesis, is an integral membrane protein of the endoplasmic reticulum (ER). In both humans and yeast, HMG-R is degraded at or in the ER. The degradation of HMG-R is regulated as part of feedback control of the mevalonate pathway. Neither the mechanism of degradation nor the nature of the signals that couple the degradation of HMG-R to the mevalonate pathway is known. We have launched a genetic analysis of the degradation of HMG-R in Saccharomyces cerevisiae using a selection for mutants that are deficient in the degradation of Hmg2p, an HMG-R isozyme. The underlying genes are called HRD (pronounced "herd"), for HMG-CoA reductase degradation. So far we have discovered mutants in three genes: HRD1, HRD2, and HRD3. The sequence of the HRD2 gene is homologous to the p97 activator of the 26S proteasome. This p97 protein, also called TRAP-2, has been proposed to be a component of the mature 26S proteasome. The hrd2-1 mutant had numerous pleiotropic phenotypes expected for cells with a compromised proteasome, and these phenotypes were complemented by the human TRAP-2/p97 coding region. In contrast, HRD1 and HRD3 genes encoded previously unknown proteins predicted to be membrane bound. The Hrd3p protein was homologous to the Caenorhabditis elegans sel-1 protein, a negative regulator of at least two different membrane proteins, and contained an HRD3 motif shared with several other proteins. Hrd1p had no full-length homologues, but contained an H2 ring finger motif. These data suggested a model of ER protein degradation in which the Hrd1p and Hrd3p proteins conspire to deliver HMG-R to the 26S proteasome. Moreover, our results lend in vivo support to the proposed role of the p97/TRAP-2/Hrd2p protein as a functionally important component of the 26S proteasome. Because the HRD genes were required for the degradation of both regulated and unregulated substrates of ER degradation, the HRD genes are the agents of HMG-R degradation but not the regulators of that degradation.

Download Full-text

Identification of signal sequence binding proteins integrated into the rough endoplasmic reticulum membrane

Biochemical Journal ◽

10.1042/bj2420767 ◽

1987 ◽

Vol 242 (3) ◽

pp. 767-777 ◽

Cited By ~ 24

Author(s):

A Robinson ◽

M A Kaderbhai ◽

B M Austen

Keyword(s):

Endoplasmic Reticulum ◽

Rough Endoplasmic Reticulum ◽

Signal Peptide ◽

Signal Sequence ◽

Secretory Protein ◽

Phospholipid Bilayer ◽

Signal Peptidase ◽

Secretory Proteins ◽

Endoplasmic Reticulum Membrane

An azidophenacyl derivative of a chemically synthesized consensus signal peptide has been prepared. The peptide, when photoactivated in the presence of rough or high-salt-stripped microsomes from pancreas, leads to inhibition of their activity in cotranslational processing of secretory pre-proteins translated from their mRNA in vitro. The peptide binds specifically with high affinity to components in the microsomal membranes from pancreas and liver, and photoreaction of a radioactive form of the azidophenacyl derivative leads to covalent linkage to yield two closely related radiolabelled proteins of Mr about 45,000. These proteins are integrated into the membrane, with large 30,000-Mr domains embedded into the phospholipid bilayer to which the signal peptide binds. A smaller, endopeptidase-sensitive, domain is exposed on the cytoplasmic surface of the microsomal vesicles. The specificity and selectivity of the binding of azidophenacyl-derivatized consensus signal peptide was demonstrated by concentration-dependent inhibition of photolabelling by the ‘cold’ synthetic consensus signal peptide and by a natural internal signal sequence cleaved and isolated from ovalbumin. The properties of the labelled 45,000-Mr protein-signal peptide complexes, i.e. mass, pI, ease of dissociation from the membrane by detergent or salts and immunological properties, distinguish them from other proteins, e.g. subunits of signal recognition particle, docking protein and signal peptidase, already known to be involved in targetting and processing of nascent secretory proteins at the rough endoplasmic reticulum membrane. Although the 45,000-Mr signal peptide binding protein displays properties similar to those of the signal peptidase, a component of the endoplasmic reticulum, the azido-derivatized consensus signal peptide does not interact with it. It is proposed that the endoplasmic reticulum proteins with which the azidophenacyl-derivatized consensus signal peptide interacts to yield the 45,000-Mr adducts may act as receptors for signals in nascent secretory pre-proteins in transduction of changes in the endoplasmic reticulum which bring about translocation of secretory protein across the membrane.

Download Full-text