CO-TRANSLATIONAL AND POSTTRANSLATIONAL TRANSLOCATION MECHANISMS DIRECT CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR N-TERMINUS TRANSMEMBRANE ASSEMBLY

Transmembrane topology of most eukaryotic polytopic proteins is establ ished cotranslationally at the endo plasmic reticulum membrane through the action of alternating signal and stop transfer sequences. Here we demonstrate that the cystic fibrosis transmembrane conductance regula tor (CFTR) achieves its N terminus topology through a variation of thi s mechanism that involves both co-and posttranslational translocation events. Using a series of defined chimeric and truncated proteins expr essed in a reticulocyte lysate system, we have identified two topogeni c determinants encoded within the first (TM1) and second (TM2) membran e-spanning segments of CFTR. Each sequence independently (i) directed endoplasmic reticulum targeting, (ii) translocated appropriate flankin g residues, and (iii) achieved its proper membrane-spanning orientatio n. Signal sequence activity of TM1, however, was inefficient due to th e presence of two charged residues, Glu(92) and Lys(95), located withi n its hydrophobic core, As a result, TM1 was able to direct correct to pology 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 direc t CFTR N terminus topology through a ribosome dependent posttranslatio nal mechanism. Mutating charged residues Glu(92) and Lys(95) to alanin e improved TM1 signal sequence activity as well as the ability of TM1 to independently direct CFTR N terminus topology, Thus, a single funct ional signal sequence in either the first or second TM segment was suf ficient for directing proper CFTR topology. These results identify two distinct and redundant translocation pathways for CFTR N terminus tra nsmembrane assembly and support a model in which TM2 functions to ensu re correct topology of CFTR chains that fail to translocate via TM1. T his novel arrangement of topogenic information provides an alternative to conventional cotranslational pathways of polytopic protein biogene sis.