A Few Positively Charged Residues Slow Movement of a Polypeptide Chain across the Endoplasmic Reticulum Membrane

Abstract

Many polypeptide chains are translocated across and integrated into the endoplasmic reticulum membrane through protein-conducting channels. During the process, amino acid sequences of translocating polypeptide chains are scanned by the channels and classified to be retained in the membrane or translocated into the lumen. We established an experimental system with which the kinetic effect of each amino acid residue on the polypeptide chain movement can be analyzed with a time resolution of tens of seconds. Positive charges greatly slow movement; only two lysine residues caused a remarkable slow down, and their effects were additive. The lysine residue was more effective than arginine. In contrast, clusters comprising three residues of each of the other 18 amino acids had little effect on chain movement. We also demonstrated that a four lysine cluster can exert the effect after being fully exposed from the ribosome. We concluded that as few as two to three residues of positively charged amino acids can slow the movement of the nascent polypeptide chain across the endoplasmic reticulum membrane. This effect provides a fundamental basis of the topogenic function of positively charged amino acids.

Stability and flexibility of marginally hydrophobic–segment stalling at the endoplasmic reticulum translocon

“…In the presence of 6 Lys (6K) residues just downstream of the segments, however, even a 2S segment (here we refer to each segment by a number and the one-letter abbreviation of the introduced amino acid) was efficient for the arrest because the 6K promoted translocation arrest of the preceding H-segments, presumably by interaction within the ribosome ( Lu and Deutsch, 2008 ) and/or any factors around the ribosome–translocon junction ( Fujita et al. , 2011 ; Yamagishi et al. , 2014 ).…”

“…, 2013 ), positively charged residues ( Fujita et al. , 2011 ; Yamagishi et al. , 2014 ), and their cooperative actions ( Fujita et al.…”

The Pathway of a Transmembrane Helix Insertion into the Membrane Assisted by Sec61α Channel

“…In addition, a short helix called a plug, which is located below the pore ring, may occlude the exit to prevent the polypeptide from passing through the bilayer if it is a membrane protein. Among the ten TMHs, TM2 and TM7 form a lateral gate that may open to allow the polypeptide to enter the bilayer. − Many experimental and theoretical studies have explored the translocation assisted by Sec61/SecY from structure change, insertion free energy, and kinetics . However, some details of the mechanism are still unclear because the whole process of translocation is extremely complex.…”

“…8−10 Many experimental and theoretical studies have explored the translocation assisted by Sec61/SecY from structure change, 11 insertion free energy, 12 and kinetics. 13 However, some details of the mechanism are still unclear because the whole process of translocation is extremely complex. A recent review paper summarizes the research progress of TMH insertion and folding.…”