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 a cquire their transmembrane (TM) topology in the endoplasmic reticulum (ER), AQP4 acquires each of its six TM segments via cotranslational translocatio n events, whereas AQP1 is initially synthesized with four TM segments and s ubsequently converted into a six membrane-spanning topology, To identify se quence determinants responsible for these pathways, peptide segments from A QP1 and AQP4 were systematically exchanged, Chimeric proteins were then tru ncated, fused to a C-terminal translocation reporter, and topology was anal yzed 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 tw o 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 r egion was identified within the TM3-4 peptide loop that enabled AQP4-TM3 bu t not AQP1-TM3 to reinitiate translocation and cotranslationally span the m embrane. Based on these findings, it was possible to convert AQP1 into a co translational 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, Int erestingly, each of these substitutions disrupted water channel activity. T hese data thus establish the structural basis for different AQP folding pat hways and provide evidence that variations in cotranslational folding enabl e polytopic proteins to acquire and/or maintain primary sequence determinan ts necessary for function.