Dept. of Physiology & Pharmacology
Oregon Health & Science University
Aquaporins (AQPs) are small (~29 kDa) hydrophobic proteins that belong to the Major Intrinsic Protein (MIP) family of glycerol and water channels. AQPs are 6-transmembranespanning proteins with two additional helical regions flanked by conserved NPA motifs that fold inward within the plane of the membrane to create a selectivity filter. They are ubiquitously expressed and best characterized within the kidney water reabsorption system. Four AQPs are responsible for water reabsorption in the nephron; AQPl in the proximal tubule and AQP2, AQP3, and AQP4 in the collecting duct. Over 30 mutations in AQP2 have been shown to cause hereditary nephrogenic diabetes insipidus (NDI), a disease characterized by the inability to produce concentrated urine. AQPs traffic as homotetramers and transport water passively across the plasma membrane according to concentration gradient. While the recently determined crystal structure of AQP family members has lead to a more complete understanding of the architecture of the AQP water channel and pore, very little is known about the early folding events preceding the formation of a functional channel. For this reason my studies have focused on investigating the early steps of AQP topogenesis, processing and folding at the ER and how these events may ultimately affect trafficking and function. AQPs and other polytopic membrane proteins are synthesized and oriented in the ER by the ribosome-translocon complex. N-linked glycosylation occurs cotranslationally as a protein is translocated into the ER. Only a subset of AQP2 is glycosylated (~20-30%). Interestingly, we found that while glycosylation had no apparent effect on WT protein it markedly stabilized the AQP2 trafficking mutants Tl26M, Al47T, C181W and Rl87C in comparison to the nonglycosylated population of protein (~25 h in comparison to~5 h). Therefore Nlinked glycans appear have a general mechanism to compensate in some general way for folding defects throughout the protein. Knowledge of whether diverse expression systems recognize topogenic information similarly is crucial for a complete understanding of the biogenesis process. Using a systematic comparison of oocyte and mammalian cell systems, we demonstrated that AQP1 's unique topogenesis whereby it is initially synthesized as a 4-TM protein, which is then converted to the mature 6-spanning channel is conserved across systems, and that the information encoded with the nascent chain itself is primarily responsible for a protein's orientation within the lipid bilayer. Further, these studies also showed that truncated proteins lacking C-terminal information could generate multiple biogenesis intermediates that reflect different steps in early protein biogenesis. These intermediates exhibit very different stabilities depending on the stage of protein biosynthesis, and this should be taken into consideration when using truncated proteins to study topogenesis. Previous work has shown that two polar residues in AQP1 TM2, Asn49 andLys51, were necessary for TM2 to slip into the ER lumen. We demonstrate that these two residues specifically interact with a charged residue in TM5, Asp 185. Through intramolecular interactions, Asn49 and Asp185 are necessary for monomer folding, whereas intermolecular interactions between Lys51 and Asp 185 help to stabilize the tetramer. These two residues are necessary to compensate for the charged Asp185, and thus AQP1 's unique biogenesis is a consequence of this requirement. Importantly, these studies outline the complex relationship between topogenesis events, tertiary folding and oligomerization of a polytopic membrane protein. Together the results presented in this thesis advance our basic knowledge of early events of AQP synthesis and folding and may well be applied to other multi-spanning proteins.
School of Medicine
Buck, Teresa M., "Molecular analysis of early aquaporin biogenesis and folding events" (2006). Scholar Archive. 821.