Biological insertion of computationally designed short transmembrane segments

The great majority of helical membrane proteins are inserted co-translationally into the ER membrane through a continuous ribosome-translocon channel. The efficiency of membrane insertion depends on transmembrane (TM) helix amino acid composition, the helix length and the position of the amino acids within the helix. In this work, we conducted a computational analysis of the composition and location of amino acids in transmembrane helices found in membrane proteins of known structure to obtain an extensive set of designed polypeptide segments with naturally occurring amino acid distributions. Then, using an in vitro translation system in the presence of biological membranes, we experimental…

Ala-insertion scanning mutagenesis of the glycophorin A transmembrane helix

Alanine insertions into the glycophorinA transmembrane helix are found to disrupthelix-helix dimerization in a way thatis fully consistentwith earlier saturation mutagenesis datas,uggesting that Ala-insertion scanning can be used to rapidly map the approximate locatiofn structurally and/or functionally importantsegments in trans-membrane helices

In eukaryotic cells, proteins are translocated across the ER membrane through a continuous ribosome-translocon channel. It is unclear to what extent proteins can fold already within the ribosome-translocon channel, and previous studies suggest that only a limited degree of folding (such as the formation of isolated α-helices) may be possible within the ribosome. We have previously shown that the conformation of nascent polypeptide chains in transit through the ribosome-translocon complex can be probed by measuring the number of residues required to span the distance between the ribosomal P-site and the lumenally disposed active site of the oligosaccharyl transferase enzyme (J. Biol. Chem 27…

Insertion and Topology of a Plant Viral Movement Protein in the Endoplasmic Reticulum Membrane

Virus-encoded movement proteins (MPs) mediate cell-to-cell spread of viral RNA through plant membranous intercellular connections, the plasmodesmata. The molecular pathway by which MPs interact with viral genomes and target plasmodesmata channels is largely unknown. The 9-kDa MP from carnation mottle carmovirus (CarMV) contains two potential transmembrane domains. To explore the possibility that this protein is in fact an intrinsic membrane protein, we have investigated its insertion into the endoplasmic reticulum membrane. By using in vitro translation in the presence of dog pancreas microsomes, we demonstrate that CarMV p9 inserts into the endoplasmic reticulum without the aid of any addi…

Transmembrane but not soluble helices fold inside the ribosome tunnel

Integral membrane proteins are assembled into the ER membrane via a continuous ribosome-translocon channel. The hydrophobicity and thickness of the core of the membrane bilayer leads to the expectation that transmembrane (TM) segments minimize the cost of harbouring polar polypeptide backbones by adopting a regular pattern of hydrogen bonds to form α-helices before integration. Co-translational folding of nascent chains into an α-helical conformation in the ribosomal tunnel has been demonstrated previously, but the features governing this folding are not well understood. In particular, little is known about what features influence the propensity to acquire α-helical structure in the ribosom…

Distant downstream sequence determinants can control N-tail translocation during protein insertion into the endoplasmic reticulum membrane.

We have studied the membrane insertion of ProW, an Escherichia coli inner membrane protein with seven transmembrane segments and a large periplasmic N-terminal tail, into endoplasmic reticulum (ER)-derived dog pancreas microsomes. Strikingly, significant levels of N-tail translocation is seen only when a minimum of four of the transmembrane segments are present; for constructs with fewer transmembrane segments, the N-tail remains mostly nontranslocated and the majority of the molecules adopt an 'inverted' topology where normally nontranslocated parts are translocated and vice versa. N-tail translocation can also be promoted by shortening of the N-tail and by the addition of positively charg…