Biogenesis and transmembrane topology of the CHIP28 water channel at the endoplasmic reticulum.

112

106

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.

Section: Cftr-tm1 Functions As An Inefficient Signal Sequence Tomentioning

confidence: 81%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

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

Lu¹,

Xiong²,

Helm

et al. 1998

112

106

“…4B) (4,37,39,40,42). The cassette contains an N-terminal cleaved signal sequence followed by two passenger domains derived from ␣-globin (N-terminal to TM8) and prolactin (C-terminal to TM8).…”

Section: Resultsmentioning

confidence: 99%

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

Carveth

Buck

Anthony

et al. 2002

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...

“…For instance, it has been shown for AQP1 that conformational changes and rearrangements take place during insertion in the endoplasmic reticulum (ER), but whether insertion occurs cotranslationally or post-translationally has become a controversial issue (10,11). In addition, with the exception of AQP4 (12), the sorting signals required for transport of AQPs between intracellular compartments remain to be defined.…”

mentioning

confidence: 99%

Glycosylation Is Important for Cell Surface Expression of the Water Channel Aquaporin-2 but Is Not Essential for Tetramerization in the Endoplasmic Reticulum

Hendriks¹,

Koudijs²,

Balkom³

et al. 2004

137

115

Aquaporin-2 (AQP2) is a pore-forming protein that is required for regulated reabsorption of water from urine. Mutations in AQP2 lead to nephrogenic diabetes insipidus, a disorder in which functional AQP2 is not expressed on the apical cell surface of kidney collecting duct principal cells. The mechanisms and pathways directing AQP2 from the endoplasmic reticulum to the Golgi complex and beyond have not been defined. We found that ϳ25% of newly synthesized AQP2 is glycosylated. Nonglycosylated and complex-glycosylated wildtype AQP2 are stable proteins with a half-life of 6 -12 h and are both detectable on the cell surface. We show that AQP2 forms tetramers in the endoplasmic reticulum during or very early after synthesis and reaches the Golgi complex in 1-1.5 h. We also report that glycosylation is neither essential for tetramerization nor for transport from the endoplasmic reticulum to the Golgi complex. Instead, the N-linked glycan is important for exit from the Golgi complex and sorting of AQP2 to the plasma membrane. These results are important for understanding the molecular mechanisms responsible for the intracellular retention of AQP2 in nephrogenic diabetes insipidus.