scholarly journals Biogenesis and transmembrane topology of the CHIP28 water channel at the endoplasmic reticulum.

1994 ◽  
Vol 125 (4) ◽  
pp. 803-815 ◽  
Author(s):  
W R Skach ◽  
L B Shi ◽  
M C Calayag ◽  
A Frigeri ◽  
V R Lingappa ◽  
...  
Keyword(s):  
Structural Model ◽  
Signal Sequence ◽  
Water Channel ◽  
Integral Membrane Protein ◽  
Coding Region ◽  
Hydrophobic Region ◽  
Transmembrane Topology ◽  
Nascent Chain ◽  
Membrane Spanning ◽  
Er Membrane

CHIP28 is a 28-kD hydrophobic integral membrane protein that functions as a water channel in erythrocytes and renal tubule epithelial cell membranes. We examined the transmembrane topology of CHIP28 in the ER by engineering a reporter of translocation (derived from bovine prolactin) into nine sequential sites in the CHIP28 coding region. The resulting chimeras were expressed in Xenopus oocytes, and the topology of the reporter with respect to the ER membrane was determined by protease sensitivity. We found that although hydropathy analysis predicted up to seven potential transmembrane regions, CHIP28 spanned the membrane only four times. Two putative transmembrane helices, residues 52-68 and 143-157, reside on the lumenal and cytosolic surfaces of the ER membrane, respectively. Topology derived from these chimeric proteins was supported by cell-free translation of five truncated CHIP28 cDNAs, by N-linked glycosylation at an engineered consensus site in native CHIP28 (residue His69), and by epitope tagging of the CHIP28 amino terminus. Defined protein chimeras were used to identify internal sequences that direct events of CHIP28 topogenesis. A signal sequence located within the first 52 residues initiated nascent chain translocation into the ER lumen. A stop transfer sequence located in the hydrophobic region from residues 90-120 terminated ongoing translocation. A second internal signal sequence, residues 155-186, reinitiated translocation of a COOH-terminal domain (residues 186-210) into the ER lumen. Integration of the nascent chain into the ER membrane occurred after synthesis of 107 residues and required the presence of two membrane-spanning regions. From this data, we propose a structural model for CHIP28 at the ER membrane in which four membrane-spanning alpha-helices form a central aqueous channel through the lipid bilayer and create a pathway for water transport.

scholarly journals Stage- and ribosome-specific alterations in nascent chain-Sec61p interactions accompany translocation across the ER membrane.

1995 ◽  
Vol 129 (4) ◽  
pp. 957-970 ◽  
Author(s):  
C V Nicchitta ◽  
E C Murphy ◽  
R Haynes ◽  
G S Shelness
Keyword(s):  
Protein Translocation ◽  
Signal Sequence ◽  
Near Neighbor ◽  
Cross Linking ◽  
Dramatic Reduction ◽  
Wild Type ◽  
Early Stages ◽  
Nascent Chain ◽  
Chemical Cross Linking ◽  
Er Membrane

Near-neighbor interactions between translocating nascent chains and Sec61p were investigated by chemical cross-linking. At stages of translocation before signal sequence cleavage, nascent chains could be cross-linked to Sec61p at high (60-80%) efficiencies. Cross-linking occurred through the signal sequence and the mature portion of wild-type and signal cleavage mutant nascent chains. At later stages of translocation, as represented through truncated translocation intermediates, cross-linking to Sec61p was markedly reduced. Dissociation of the ribosome into its large and small subunits after assembly of the precursor into the translocon, but before cross-linking, resulted in a dramatic reduction in subsequent cross-linking yield, indicating that at early stages of translocation, nascent chain-Sec61p interactions are in part mediated through interactions of the ribosome with components of the ER membrane, such as Sec61p. Dissociation of the ribosome was, however, without effect on subsequent translocation. These results are discussed with respect to a model in which Sec61p performs a function essential for the initiation of protein translocation.

scholarly journals Transient translocation of the cytoplasmic (endo) domain of a type I membrane glycoprotein into cellular membranes.

1993 ◽  
Vol 120 (4) ◽  
pp. 877-883 ◽  
Author(s):  
N Liu ◽  
D T Brown
Keyword(s):  
Amino Acids ◽  
Membrane Protein ◽  
Signal Sequence ◽  
Attachment Site ◽  
Integral Membrane Protein ◽  
Carboxyl Terminus ◽  
Type I ◽  
E2 Glycoprotein ◽  
Typical Type ◽  
Membrane Spanning

The E2 glycoprotein of the alphavirus Sindbis is a typical type I membrane protein with a single membrane spanning domain and a cytoplasmic tail (endo domain) containing 33 amino acids. The carboxyl terminal domain of the tail has been implicated as (a) attachment site for nucleocapsid protein, and (b) signal sequence for integration of the other alpha-virus membrane proteins 6K and E1. These two functions require that the carboxyl terminus be exposed in the cell cytoplasm (a) and exposed in the lumen of the endoplasmic reticulum (b). We have investigated the orientation of this glycoprotein domain with respect to cell membranes by substituting a tyrosine for the normally occurring serine, four amino acids upstream of the carboxyl terminus. Using radioiodination of this tyrosine as an indication of the exposure of the glycoprotein tail, we have provided evidence that this domain is initially translocated into a membrane and is returned to the cytoplasm after export from the ER. This is the first demonstration of such a transient translocation of a single domain of an integral membrane protein and this rearrangement explains some important aspects of alphavirus assembly.

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 Signal sequence-dependent function of the TRAM protein during early phases of protein transport across the endoplasmic reticulum membrane.

1996 ◽  
Vol 134 (1) ◽  
pp. 25-35 ◽  
Author(s):  
S Voigt ◽  
B Jungnickel ◽  
E Hartmann ◽  
T A Rapoport
Keyword(s):  
Protein Transport ◽  
Signal Sequence ◽  
Chain Complex ◽  
Secretory Proteins ◽  
Endoplasmic Reticulum Membrane ◽  
Dependent Manner ◽  
Hydrophobic Core ◽  
Sequence Dependent ◽  
Nascent Chain ◽  
Er Membrane

Cotranslational translocation of proteins across the mammalian ER membrane involves, in addition to the signal recognition particle receptor and the Sec61p complex, the translocating chain-associating membrane (TRAM) protein, the function of which is still poorly understood. Using reconstituted proteoliposomes, we show here that the translocation of most, but not all, secretory proteins requires the function of TRAM. Experiments with hybrid proteins demonstrate that the structure of the signal sequence determines whether or not TRAM is needed. Features that distinguish TRAM-dependent and -independent signal sequences include the length of their charged, NH2-terminal region and the structure of their hydrophobic core. In cases where TRAM is required for translocation, it is not needed for the initial interaction of the ribosome/nascent chain complex with the ER membrane but for a subsequent step inside the membrane in which the nascent chain is inserted into the translocation site in a protease-resistant manner. Thus, TRAM functions in a signal sequence-dependent manner at a critical, early phase of the translocation process.

scholarly journals Transport of the Intracisternal A-Type Particle Gag Polyprotein to the Endoplasmic Reticulum Is Mediated by the Signal Recognition Particle

2003 ◽  
Vol 77 (11) ◽  
pp. 6293-6304 ◽  
Author(s):  
Frauke Fehrmann ◽  
Martin Jung ◽  
Richard Zimmermann ◽  
Hans-Georg Kräusslich
Keyword(s):  
Endoplasmic Reticulum ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Endogenous Retroviruses ◽  
In Vitro Translation ◽  
Signal Recognition ◽  
Hydrophobic Region ◽  
Gag Polyprotein ◽  
Er Targeting ◽  
Er Membrane

ABSTRACT Intracisternal A-type particles (IAP) are defective endogenous retroviruses that accumulate in the endoplasmic reticulum (ER) of rodent cells. The enveloped particles are produced by assembly and budding of IAP Gag polyproteins at the ER membrane. In this study, we analyzed the specific ER transport of the Gag polyprotein of the IAP element MIA14. To this end, we performed in vitro translation of Gag in the presence of microsomal membranes or synthetic proteoliposomes followed by membrane sedimentation or flotation. ER binding of IAP Gag occurred mostly cotranslationally, and Gag polyproteins interacted specifically with proteoliposomes containing only signal recognition particle (SRP) receptor and the Sec61p complex, which form the minimal ER translocation apparatus. The direct participation of SRP in ER targeting of IAP Gag was demonstrated in cross-linking and immunoprecipitation experiments. The IAP polyprotein was not translocated into the ER; it was found to be tightly associated with the cytoplasmic side of the ER membrane but did not behave as an integral membrane protein. Substituting the functional signal peptide of preprolactin for the hydrophobic sequence at the N terminus of IAP Gag also did not result in translocation of the chimeric protein into the ER lumen, and grafting the IAP hydrophobic sequence onto preprolactin failed to yield luminal transport as well. These results suggest that the N-terminal hydrophobic region of the IAP Gag polyprotein functions as a transport signal which mediates SRP-dependent ER targeting, but polyprotein translocation or integration into the membrane is prevented by the signal sequence itself and by additional regions of Gag.

scholarly journals Biosynthesis of the dystonia-associated AAA+ ATPase torsinA at the endoplasmic reticulum

2006 ◽  
Vol 401 (2) ◽  
pp. 607-612 ◽  
Author(s):  
Anna C. Callan ◽  
Sandra Bunning ◽  
Owen T. Jones ◽  
Stephen High ◽  
Eileithyia Swanton
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Protein ◽  
Transmembrane Domain ◽  
Signal Sequence ◽  
Integral Membrane Protein ◽  
Signal Peptidase ◽  
Aaa Atpase ◽  
C Terminus ◽  
Membrane Spanning ◽  
Aaa Atpases

TorsinA is a widely expressed AAA+ (ATPases associated with various cellular activities) ATPase of unknown function. Previous studies have described torsinA as a type II protein with a cleavable signal sequence, a single membrane spanning domain, and its C-terminus located in the ER (endoplasmic reticulum) lumen. However, in the present study we show that torsinA is not in fact an integral membrane protein. Instead we find that the mature protein associates peripherally with the ER membrane, most likely through an interaction with an integral membrane protein. Consistent with this model, we provide evidence that the signal peptidase complex cleaves the signal sequence of torsinA, and we show that the region previously suggested to form a transmembrane domain is translocated into the lumen of the ER. The finding that torsinA is a peripheral, and not an integral membrane protein as previously thought, has important implications for understanding the function of this novel ATPase.

scholarly journals Requirement of the N-Terminal Region of Orthobunyavirus Nonstructural Protein NSm for Virus Assembly and Morphogenesis

2006 ◽  
Vol 80 (16) ◽  
pp. 8089-8099 ◽  
Author(s):  
Xiaohong Shi ◽  
Alain Kohl ◽  
Vincent H. J. Léonard ◽  
Ping Li ◽  
Angela McLees ◽  
...  
Keyword(s):  
Reverse Genetics ◽  
Fluorescent Protein ◽  
Virus Assembly ◽  
Signal Sequence ◽  
Nonstructural Protein ◽  
Intracellular Localization ◽  
Integral Membrane Protein ◽  
Terminal Region ◽  
Coding Region ◽  
Hydrophobic Domain

ABSTRACT The nonstructural protein NSm of Bunyamwera virus (BUNV), the prototype of the Bunyaviridae family, is encoded by the M segment in a polyprotein precursor, along with the virion glycoproteins, in the order Gn-NSm-Gc. As little is known of its function, we examined the intracellular localization, membrane integrality, and topology of NSm and its role in virus replication. We confirmed that NSm is an integral membrane protein and that it localizes in the Golgi complex, together with Gn and Gc. Coimmunoprecipitation assays and yeast two-hybrid analysis demonstrated that NSm was able to interact with other viral proteins. NSm is predicted to contain three hydrophobic (I, III, and V) and two nonhydrophobic (II and IV) domains. The N-terminal nonhydrophobic domain II was found in the lumen of an intracellular compartment. A novel BUNV assembly assay was developed to monitor the formation of infectious virus-like-particles (VLPs). Using this assay, we showed that deletions of either the complete NSm coding region or domains I, II, and V individually seriously compromised VLP production. Consistently, we were unable to rescue viable viruses by reverse genetics from cDNA constructs that contained the same deletions. However, we could generate mutant BUNV with deletions in NSm domains III and IV and also a recombinant virus with the green fluorescent protein open reading frame inserted into NSm domain IV. The mutant viruses displayed differences in their growth properties. Overall, our data showed that the N-terminal region of NSm, which includes domain I and part of domain II, is required for virus assembly and that the C-terminal hydrophobic domain V may function as an internal signal sequence for the Gc glycoprotein.

scholarly journals Binding of Signal Recognition Particle Gives Ribosome/Nascent Chain Complexes a Competitive Advantage in Endoplasmic Reticulum Membrane Interaction

1998 ◽  
Vol 9 (1) ◽  
pp. 103-115 ◽  
Author(s):  
Andrea Neuhof ◽  
Melissa M. Rolls ◽  
Berit Jungnickel ◽  
Kai-Uwe Kalies ◽  
Tom A. Rapoport
Keyword(s):  
Endoplasmic Reticulum ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Membrane Binding ◽  
Membrane Targeting ◽  
Signal Recognition ◽  
Nascent Polypeptide ◽  
Nascent Chain ◽  
Cytosolic Factors ◽  
Er Membrane

Most secretory and membrane proteins are sorted by signal sequences to the endoplasmic reticulum (ER) membrane early during their synthesis. Targeting of the ribosome-nascent chain complex (RNC) involves the binding of the signal sequence to the signal recognition particle (SRP), followed by an interaction of ribosome-bound SRP with the SRP receptor. However, ribosomes can also independently bind to the ER translocation channel formed by the Sec61p complex. To explain the specificity of membrane targeting, it has therefore been proposed that nascent polypeptide-associated complex functions as a cytosolic inhibitor of signal sequence- and SRP-independent ribosome binding to the ER membrane. We report here that SRP-independent binding of RNCs to the ER membrane can occur in the presence of all cytosolic factors, including nascent polypeptide-associated complex. Nontranslating ribosomes competitively inhibit SRP-independent membrane binding of RNCs but have no effect when SRP is bound to the RNCs. The protective effect of SRP against ribosome competition depends on a functional signal sequence in the nascent chain and is also observed with reconstituted proteoliposomes containing only the Sec61p complex and the SRP receptor. We conclude that cytosolic factors do not prevent the membrane binding of ribosomes. Instead, specific ribosome targeting to the Sec61p complex is provided by the binding of SRP to RNCs, followed by an interaction with the SRP receptor, which gives RNC–SRP complexes a selective advantage in membrane targeting over nontranslating ribosomes.

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