Role of Ribosomes in Reinitiation of Membrane Insertion of Internal Transmembrane Segments in a Polytopic Membrane Protein†

Biochemistry ◽  
1997 ◽  
Vol 36 (38) ◽  
pp. 11437-11443 ◽  
Author(s):  
Changsen Wang ◽  
Mingang Chen ◽  
Ernest Han ◽  
Jian-Ting Zhang
Keyword(s):  
Membrane Protein ◽  
Membrane Insertion ◽  
Transmembrane Segments ◽  
Polytopic Membrane Protein

scholarly journals The Role of the Protein-Conducting Channel in the Membrane Insertion of Transmembrane Segments

2010 ◽  
Vol 98 (3) ◽  
pp. 224a
Author(s):  
James C. Gumbart ◽  
Christophe Chipot ◽  
Klaus Schulten
Keyword(s):  
Membrane Insertion ◽  
Conducting Channel ◽  
Transmembrane Segments

scholarly journals Glycosylation of multiple extracytosolic loops in Band 3, a model polytopic membrane protein

1996 ◽  
Vol 318 (2) ◽  
pp. 645-648 ◽  
Author(s):  
Lisa Y TAM ◽  
Carolina LANDOLT-MARTICORENA ◽  
Reinhart A. F. REITHMEIER
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Band 3 ◽  
Site Directed Mutagenesis ◽  
Acceptor Site ◽  
Oligosaccharyl Transferase ◽  
Transmembrane Segments ◽  
Free Translation ◽  
Reticulocyte Lysates ◽  
Polytopic Membrane Protein

N-glycosylated sites in polytopic membrane proteins are usually localized to single extracytosolic (EC) loops containing more than 30 residues [Landolt-Marticorena and Reithmeier (1994) Biochem. J. 302, 253–260]. This may be due to a biosynthetic restriction whereby only a single loop of nascent polypeptide is available to the oligosaccharyl transferase in the lumen of the endoplasmic reticulum. To test this hypothesis, two types of N-glycosylation mutants were constructed using Band 3, a polytopic membrane protein that contains up to 14 transmembrane segments and a single endogenous site of N-glycosylation at Asn-642 in EC loop 4. In the first set of mutants, an additional N-glycosylation acceptor site (Asn-Xaa-Ser/Thr) was constructed by site-directed mutagenesis in EC loop 3, with or without retention of the endogenous site. In the second set of mutants, EC loop 4 was duplicated and inserted into EC loop 2, again with or without retention of the endogenous site. Cell-free translation experiments using reticulocyte lysates showed that microsomes were able to N-glycosylate multiple EC loops in these Band 3 mutants. The acceptor site in EC loop 3 was poorly N-glycosylated, probably due to the suboptimal size (25 residues) of this EC loop. The localization of N-glycosylation sites to single EC loops in multi-span membrane proteins is probably due to the absence of suitably positioned acceptor sites on multiple loops.

scholarly journals Mapping the Ends of Transmembrane Segments in a Polytopic Membrane Protein

1997 ◽  
Vol 272 (29) ◽  
pp. 18325-18332 ◽  
Author(s):  
Milka Popov ◽  
Lisa Y. Tam ◽  
Jing Li ◽  
Reinhart A. F. Reithmeier
Keyword(s):  
Membrane Protein ◽  
Transmembrane Segments ◽  
Polytopic Membrane Protein

scholarly journals Visualization of a polytopic membrane protein during SecY-mediated membrane insertion

2014 ◽  
Vol 5 (1) ◽  
Author(s):  
Lukas Bischoff ◽  
Stephan Wickles ◽  
Otto Berninghausen ◽  
Eli O. van der Sluis ◽  
Roland Beckmann
Keyword(s):  
Membrane Protein ◽  
Membrane Insertion ◽  
Polytopic Membrane Protein

scholarly journals Targeting of a polytopic membrane protein to the inner envelope membrane of chloroplasts in vivo involves multiple transmembrane segments

2014 ◽  
Vol 65 (18) ◽  
pp. 5257-5265 ◽  
Author(s):  
Kumiko Okawa ◽  
Hitoshi Inoue ◽  
Fumi Adachi ◽  
Katsuhiro Nakayama ◽  
Yasuko Ito-Inaba ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Envelope Membrane ◽  
Transmembrane Segments ◽  
Polytopic Membrane Protein ◽  
Inner Envelope

scholarly journals Dissection of De Novo Membrane Insertion Activities of Internal Transmembrane Segments of ATP-Binding-Cassette Transporters: Toward Understanding Topological Rules for Membrane Assembly of Polytopic Membrane Proteins

1998 ◽  
Vol 9 (4) ◽  
pp. 853-863 ◽  
Author(s):  
Jian-Ting Zhang ◽  
Mingang Chen ◽  
Ernest Han ◽  
Changsen Wang
Keyword(s):  
Membrane Proteins ◽  
De Novo ◽  
Chinese Hamster ◽  
Membrane Insertion ◽  
Strong Activity ◽  
Membrane Assembly ◽  
Transmembrane Segments ◽  
Model Protein ◽  
Tm Domain ◽  
Polytopic Membrane Protein

The membrane assembly of polytopic membrane proteins is a complicated process. Using Chinese hamster P-glycoprotein (Pgp) as a model protein, we investigated this process previously and found that Pgp expresses more than one topology. One of the variations occurs at the transmembrane (TM) domain including TM3 and TM4: TM4 inserts into membranes in an Nin-Cout rather than the predicted Nout-Cin orientation, and TM3 is in cytoplasm rather than the predicted Nin-Coutorientation in the membrane. It is possible that TM4 has a strong activity to initiate the Nin-Cout membrane insertion, leaving TM3 out of the membrane. Here, we tested this hypothesis by expressing TM3 and TM4 in isolated conditions. Our results show that TM3 of Pgp does not have de novo Nin-Cout membrane insertion activity whereas TM4 initiates the Nin-Cout membrane insertion regardless of the presence of TM3. In contrast, TM3 and TM4 of another polytopic membrane protein, cystic fibrosis transmembrane conductance regulator (CFTR), have a similar level of de novo Nin-Cout membrane insertion activity and TM4 of CFTR functions only as a stop-transfer sequence in the presence of TM3. Based on these findings, we propose that 1) the membrane insertion of TM3 and TM4 of Pgp does not follow the sequential model, which predicts that TM3 initiates Nin-Cout membrane insertion whereas TM4 stops the insertion event; and 2) “leaving one TM segment out of the membrane” may be an important folding mechanism for polytopic membrane proteins, and it is regulated by the Nin-Cout membrane insertion activities of the TM segments.

scholarly journals Membrane Topology Analysis of Cyclic Glucan Synthase, a Virulence Determinant of Brucella abortus

2004 ◽  
Vol 186 (21) ◽  
pp. 7205-7213 ◽  
Author(s):  
Andrés E. Ciocchini ◽  
Mara S. Roset ◽  
Nora Iñón de Iannino ◽  
Rodolfo A. Ugalde
Keyword(s):  
Membrane Protein ◽  
Brucella Abortus ◽  
Membrane Topology ◽  
Chemical Labeling ◽  
Glucan Synthase ◽  
Transmembrane Segments ◽  
Host Interaction ◽  
Genes Encoding ◽  
Inner Membrane Protein ◽  
Polytopic Membrane Protein

ABSTRACT Brucella abortus cyclic glucan synthase (Cgs) is a 316-kDa (2,831-amino-acid) integral inner membrane protein that is responsible for the synthesis of cyclic β-1,2-glucan by a novel mechanism in which the enzyme itself acts as a protein intermediate. B. abortus Cgs uses UDP-glucose as a sugar donor and has the three enzymatic activities necessary for synthesis of the cyclic polysaccharide (i.e., initiation, elongation, and cyclization). Cyclic glucan is required in B. abortus for effective host interaction and complete expression of virulence. To gain further insight into the structure and mechanism of action of B. abortus Cgs, we studied the membrane topology of the protein using a combination of in silico predictions, a genetic approach involving the construction of fusions between the cgs gene and the genes encoding alkaline phosphatase (phoA) and β-galactosidase (lacZ), and site-directed chemical labeling of lysine residues. We found that B. abortus Cgs is a polytopic membrane protein with the amino and carboxyl termini located in the cytoplasm and with six transmembrane segments, transmembrane segments I (residues 419 to 441), II (residues 452 to 474), III (residues 819 to 841), IV (residues 847 to 869), V (residues 939 to 961), and VI (residues 968 to 990). The six transmembrane segments determine four large cytoplasmic domains and three very small periplasmic regions.

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