Co- and Posttranslational Translocation Mechanisms Direct Cystic Fibrosis Transmembrane Conductance Regulator N Terminus Transmembrane Assembly

Abstract

Transmembrane topology of most eukaryotic polytopic proteins is established cotranslationally at the endoplasmic reticulum membrane through the action of alternating signal and stop transfer sequences. Here we demonstrate that the cystic fibrosis transmembrane conductance regulator (CFTR) achieves its N terminus topology through a variation of this mechanism that involves both co-and posttranslational translocation events. Using a series of defined chimeric and truncated proteins expressed in a reticulocyte lysate system, we have identified two topogenic determinants encoded within the first (TM1) and second (TM2) membranespanning segments of CFTR. Each sequence independently (i) directed endoplasmic reticulum targeting, (ii) translocated appropriate flanking residues, and (iii) achieved its proper membrane-spanning orientation. Signal sequence activity of TM1, however, was inefficient due to the presence of two charged residues, Glu 92 and Lys 95 , located within its hydrophobic core. As a result, TM1 was able to direct correct topology for less than half of nascent CFTR chains. In contrast to TM1, TM2 signal sequence activity was both efficient and specific. Even in the absence of a functional TM1 signal sequence, TM2 was able to direct CFTR N terminus topology through a ribosome-dependent posttranslational mechanism. Mutating charged residues Glu 92 and Lys 95 to alanine improved TM1 signal sequence activity as well as the ability of TM1 to independently direct CFTR N terminus topology. Thus, a single functional signal sequence in either the first or second TM segment was sufficient for directing proper CFTR topology. These results identify two distinct and redundant translocation pathways for CFTR N terminus transmembrane assembly and support a model in which TM2 functions to ensure correct topology of CFTR chains that fail to translocate via TM1. This novel arrangement of topogenic information provides an alternative to conventional cotranslational pathways of polytopic protein biogenesis.

Expression of a functionally active human hepatic UDP‐glucuronosyltransferase (UGT1A6) lacking the N‐terminal signal sequence in the endoplasmic reticulum

“…with and n'ithout signal peptide In vitro transcription-translation in the presence of pancreas microsomal membranes is a powerful tool routinely used to study the targeting, translocation, post-translational modifications and membrane insertion of proteins [24][25][26]. This system was thus used to study the cleavage of the Nterminal signal peptide and asparagine-linked glycosylation, which are the only post-translational modifications described for UGTs as well as the membrane insertion of UGT1A6 [27].…”

Coupled Translocation Events Generate Topological Heterogeneity at the Endoplasmic Reticulum Membrane

“…Alternatively, native polytopic proteins may acquire their topology through cooperative and/or synergistic interactions between multiple topogenic determinants (Calamia and Manoil, 1992;Wilkinson et al, 1996;Lu et al, 1997). This latter process involves transient uncoupling of translocation and membrane integration events Lin and Addison, 1995), posttranslational translocation of peptide loops (Lu et al, 1997), or even posttranslational reorientation of TM helices (Wilkinson et al, 1996). Thus recent studies have added new complexities to the traditional view of polytopic protein biogenesis by demonstrating that a given TM topology may be generated through several alternate combinations of topogenic events.…”

“…In the simplest case, this process proceeds cotranslationally at the ER membrane as independent signal (anchor) and ST sequences direct sequential rounds of translocation initiation, translocation termination, and membrane integration (Rothman et al, 1988;Wessels and Spiess, 1988;Lipp et al, 1989;Shi et al, 1995). Alternatively, native polytopic proteins may acquire their topology through cooperative and/or synergistic interactions between multiple topogenic determinants (Calamia and Manoil, 1992;Wilkinson et al, 1996;Lu et al, 1997). This latter process involves transient uncoupling of translocation and membrane integration events Lin and Addison, 1995), posttranslational translocation of peptide loops (Lu et al, 1997), or even posttranslational reorientation of TM helices (Wilkinson et al, 1996).…”

Contribution of hydrophobic and electrostatic interactions to the membrane integration of the Shaker K + channel voltage sensor domain

“…This process is regulated by topogenic signals in the amino acid sequence that define the final membrane topology (16). In the simplest case the TM helices are integrated into the membrane one by one (17), but in some cases two or more TM helices in the nascent polypeptide need to interact to be able to insert efficiently (18,19). There have been several studies on the biogenesis of K v channels (20)(21)(22)(23)(24)(25), but these studies have arrived at different results, in particular as regards insertion of the voltage sensor.…”