Identification of sequence determinants that direct different intracellular folding pathways for aquaporin-1 and aquaporin-4.
Homologous aquaporin water channels utilize different folding pathways to acquire their transmembrane (TM) topology in the endoplasmic reticulum (ER). AQP4 acquires each of its six TM segments via cotranslational translocation events, whereas AQP1 is initially synthesized with four TM segments and subsequently converted into a six membrane-spanning topology. To identify sequence determinants responsible for these pathways, peptide segments from AQP1 and AQP4 were systematically exchanged. Chimeric proteins were then truncated, fused to a C-terminal translocation reporter, and topology was analyzed by protease accessibility. In each chimeric context, TM1 initiated ER targeting and translocation. However, AQP4-TM2 cotranslationally terminated translocation, while AQP1-TM2 failed to terminate translocation and passed into the ER lumen. This difference in stop transfer activity was due to two residues that altered both the length and hydrophobicity of TM2 (Asn(49) and Lys(51) in AQP1 versus Met(48) and Leu(50) in AQP4). A second peptide region was identified within the TM3-4 peptide loop that enabled AQP4-TM3 but not AQP1-TM3 to reinitiate translocation and cotranslationally span the membrane. Based on these findings, it was possible to convert AQP1 into a cotranslational biogenesis mode similar to that of AQP4 by substituting just two peptide regions at the N terminus of TM2 and the C terminus of TM3. Interestingly, each of these substitutions disrupted water channel activity. These data thus establish the structural basis for different AQP folding pathways and provide evidence that variations in cotranslational folding enable polytopic proteins to acquire and/or maintain primary sequence determinants necessary for function.
Foster
W
W
Division of Molecular Medicine, Oregon Health Sciences University, Portland, Oregon 97201, USA.
Helm
A
A
Turnbull
I
I
Gulati
H
H
Yang
B
B
Verkman
A S
AS
Skach
W R
WR
D000595:Amino Acid Sequence; D051398:Aquaporin 1; D051401:Aquaporin 4; D020346:Aquaporins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D018076:DNA, Complementary; D008969:Molecular Sequence Data; D009841:Oligonucleotides; D017510:Protein Folding; D021382:Protein Sorting Signals; D017386:Sequence Homology, Amino Acid
D051401:Aquaporin 4; D020346:Aquaporins; D018076:DNA, Complementary; D009841:Oligonucleotides; D021382:Protein Sorting Signals; D051398:Aquaporin 1