2000 Global Researcher Conference Proceeding

March 10 - 12, 2000

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Conference: 2000 Global Researcher Conference
Title: Identification of sequence determinants that direct different intracellular folding pathways for kidney aquaporins
Authors: Foster, William; Helm, Andrew; Gulati, Harnik; Verkman, Alan S.; Skach, M.D., William R.
Institutions: Oregon Health Sciences University, University of California, S.F., A.I. Dupont, University of Portland
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Abstract • Translation
Skach Homologous aquaporin water channels, AQP1 and AQP4, utilize different folding pathways to acquire their transmembrane (TM) topology in the endoplasmic reticulum (ER). The six TM segments of AQP4 are generated cotranslationally via sequential translocation events (Biochem. 34:8250, 1995), whereas AQP1 is initially synthesized with only four TM segments (J. Cell. Biol. 125:803, 1994) and subsequently converted into its mature six-spanning topology. To identify primary sequence determinants responsible for these pathways, a series of chimeric proteins was generated in which peptide segments from AQP1 and AQP4 were systematically exchanged. Chimeras were then sequentially truncated, fused to a C-terminus translocation reporter, and analyzed in rabbit reticulocyte lysate by protease accessibility. The first TM segment of both AQP1 and AQP4 efficiently directed ER targeting and polypeptide translocation. During AQP4 biogenesis, TM2 terminated translocation and spanned the membrane in its predicted orientation. In contrast, AQP1-TM2 failed to terminate translocation and passed into the ER lumen. This difference in topogenic behavior was abolished by converting two hydrophilic residues at the N-terminus of AQP1-TM2 (N49/K51) to corresponding hydrophobic residues from AQP4 (M48/L50). These changes also increased the length of AQP1-TM2 from 17-20 residues. A second region was identified within the TM3-4 peptide loop, which influenced the ability of TM3 to reinitiate translocation and cotranslationally acquire its predicted type II topology. Based on these findings, it was possible to convert AQP1 into a biogenesis pathway similar to that of AQP4 by appropriate substitution of just two peptide regions located at the N-terminus of TM2 and C-terminus of TM3. Interestingly, chimeric substitutions that altered AQP1 biogenesis events, markedly decreased water channel activity in microinjected Xenopus laevis oocytes. These results establish the structural basis for different AQP1 and AQP4 folding pathways and demonstrate that minor sequence variations can significantly influence the biogenesis and function of closely related aquaporin proteins.
By comparing the behavior of smaller and smaller pieces of protein from AQP1 and AQP4, researchers identified two regions surrounding TM2 and TM3 that were responsible for these folding differences. When these two regions from AQP4 were inserted into AQP1, AQP1 adopted a simple folding pattern indistinguishable from that of AQP4. Surprisingly, however, even small exchanges that influenced folding events completely disrupted AQP1 function. The observation that AQP1 protein regions responsible for these unusual folding events are also required for protein function begins to explain why AQP1 must utilize a more complicated folding process than AQP4. Understanding how these folding events take place for different AQP proteins is an important step in defining how mutations in AQP2, which change a single amino acid, can disrupt folding sufficiently to cause nephrogenic diabetes insipidus.