Cooperativity and Flexibility of Cystic Fibrosis Transmembrane Conductance Regulator Transmembrane Segments Participate in Membrane Localization of a Charged Residue

Abstract

Polytopic protein topology is established in the endoplasmic reticulum (ER) by sequence determinants encoded throughout the nascent polypeptide. Here we characterize 12 topogenic determinants in the cystic fibrosis transmembrane conductance regulator, and identify a novel mechanism by which a charged residue is positioned within the plane of the lipid bilayer. During cystic fibrosis transmembrane conductance regulator biogenesis, topology of the C-terminal transmembrane domain (TMs 7-12) is directed by alternating signal (TMs 7, 9, and 11) and stop transfer (TMs 8, 10, and 12) sequences. Unlike conventional stop transfer sequences, however, TM8 is unable to independently terminate translocation due to the presence of a single charged residue, Asp 924 , within the TM segment. Instead, TM8 stop transfer activity is specifically dependent on TM7, which functions both to initiate translocation and to compensate for the charged residue within TM8. Moreover, even in the presence of TM7, the N terminus of TM8 extends significantly into the ER lumen, suggesting a high degree of flexibility in establishing TM8 transmembrane boundaries. These studies demonstrate that signal sequences can markedly influence stop transfer behavior and indicate that ER translocation machinery simultaneously integrates information from multiple topogenic determinants as they are presented in rapid succession during polytopic protein biogenesis.The topology of most eukaryotic polytopic proteins is generated in the endoplasmic reticulum (ER) 1 through the collective action of sequence determinants encoded within the nascent polypeptide. These determinants encompass hydrophobic transmembrane (TM) segments that, together with their flanking residues, interact with cytosolic and ER translocation machinery to initiate and terminate translocation and integrate the polypeptide into the lipid bilayer (reviewed in Refs. 1-3). In the simplest model, topology can be established cotranslationally by alternating topogenic determinants that function as signal (anchor) and stop transfer sequences (4 -7). As the first signal sequence emerges from the ribosome, it targets the ribosome nascent-chain complex (RNC) to the ER and gates open an aqueous channel in the membrane (the Sec61 translocon) (8). Because the ribosome exit site is directly aligned with the axial pore of the translocon, newly synthesized polypeptide is cotranslationally directed into the aqueous environment of the translocon as it emerges from the ribosome (9 -11). Subsequent synthesis of a stop transfer sequence gates the translocon closed to the ER lumen, terminates translocation, and provides the growing nascent polypeptide access to the cytosol (12, 13). Through sequential iterations of these events, signal and stop transfer sequences can alternately direct the polypeptide into the ER lumen or the cytosol and thus establish topology of transmembrane segments and lumenal and cytosolic peptide loops.Not all native polytopic proteins utilize a simple cotranslational biogenesis pathway. For ex...

Biogenesis of Functional Antigenic Peptide Transporter TAP Requires Assembly of Pre-existing TAP1 with Newly Synthesized TAP2

“…different biogenesis pathways (19,20). Thus, despite their conserved topology, corresponding TM segments within the N-and C-terminal TMDs encode dissimilar topogenic information and use different translocation mechanisms to acquire their final topology (19,20). A recent publication showed that co-and post-translational steps as well as interactions between domains are required for proper folding of CFTR (21).…”

“…The bar graphs represent the average values of experiments measured in duplicate. different biogenesis pathways (19,20). Thus, despite their conserved topology, corresponding TM segments within the N-and C-terminal TMDs encode dissimilar topogenic information and use different translocation mechanisms to acquire their final topology (19,20).…”

Modulating the Folding of P-Glycoprotein and Cystic Fibrosis Transmembrane Conductance Regulator Truncation Mutants with Pharmacological Chaperones

“…The problems with proper orientation of the glycosylated loop may involve TM8. TM8 of CFTR is unusual because it has poor stop translocation properties, and removal of a negatively charged residue in TM8 (Asp 924) altered the topology of the extracellular loop between TM segments 7 and 8 (Carveth et al, 2002). Asp924 normally forms a salt bridge with Arg347 in TM6 of TMD1 (Cotten and Welsh, 1999).…”

An Energy-dependent Maturation Step Is Required for Release of the Cystic Fibrosis Transmembrane Conductance Regulator from Early Endoplasmic Reticulum Biosynthetic Machinery

“…In vitro translation systems are well suited for such analysis and have been used to define mechanisms of CFTR topogenesis (11,12,15), identify early CFTR chaperone interactions (50), and characterize CFTR degradation pathways (48,51). We previously showed that rabbit reticulocyte lysate (RRL) supplemented with ER-derived microsomal membranes faithfully reconstitutes CFTR synthesis, core glycosylation, and membrane integration (45,51).…”

“…A poorly understood, but important aspect of CFTR biogenesis is how folding and assembly of different domains are coordinated by cellular machinery in different cellular compartments. Topology of membrane-spanning domains is established during synthesis as the ribosome and nascent chain target to the ER membrane and bind a large protein-conducting channel (translocon) composed of Sec61␣ and its associated proteins (11)(12)(13)(14). As translation proceeds, internal sequences within the nascent CFTR polypeptide interact with the ribosome and translocon where they initiate polypeptide translocation into the ER lumen, orient cytosolic loops, and direct transmembrane segment integration into the lipid bilayer (12,(15)(16)(17).…”