Human aquaporin 5 (HsAQP5) facilitates the transport of water across plasma membranes and has been identified within cells of the stomach, duodenum, pancreas, airways, lungs, salivary glands, sweat glands, eyes, lacrimal glands, and the inner ear. AQP5, like AQP2, is subject to posttranslational regulation by phosphorylation, at which point it is trafficked between intracellular storage compartments and the plasma membrane. Details concerning the molecular mechanism of membrane trafficking are unknown. Here we report the x-ray structure of HsAQP5 to 2.0-Å resolution and highlight structural similarities and differences relative to other eukaryotic aquaporins. A lipid occludes the putative central pore, preventing the passage of gas or ions through the center of the tetramer. Multiple consensus phosphorylation sites are observed in the structure and their potential regulatory role is discussed. We postulate that a change in the conformation of the C terminus may arise from the phosphorylation of AQP5 and thereby signal trafficking.membrane protein ͉ trafficking ͉ crystallography ͉ water channel protein ͉ heterologous overexpression A quaporins (1) facilitate the flow of water across cellular membranes while preserving ion concentration gradients. By maintaining water homeostasis within cells, aquaporin family members play essential physiological roles within all kingdoms of life. They form a large superfamily containing both pure water channels, and channels also permeable to other small polar molecules such as glycerol (2). Thirteen human aquaporin (AQP) isoforms have been identified, and they govern a broad spectrum of physiological functions (2, 3). Examples include concentration of urine in the kidneys, release of tears and maintenance of lens transparency in the eye, maintenance of water homeostasis within the brain, the extrusion of sweat from the skin, control of glycerol concentration in fat metabolism, and facilitation of cell migration during angiogenesis.X-ray and electron diffraction studies have yielded crystal structures of mammalian AQP0 (4-6), AQP1 (7, 8), AQP2 (9), and AQP4 (10), plant SoPIP2;1 (11, 12), bacterial AQPZ (13,14), and GlpF (15), and the archaeal AQPM (16). These structures establish that phylogenetically and functionally diverse AQPs arrange as homotetramers, each protomer containing six highly conserved transmembrane (TM) ␣-helices. Two half-helices form a pseudoseventh TM helix because of the insertion of loops B and E into the membrane from opposite sides, placing both copies of the highly conserved Asn-Pro-Ala (NPA) AQP signature motif near the center of the water channel. The conserved aromatic/arginine (ar/R) constriction region imposes substrate selectivity on the channel (17). A consensus has emerged from molecular dynamics simulations regarding the mechanism of water transport and ion exclusion (18) establishing that an electrostatic potential barrier peaking at the NPA region prevents the cotranslocation of protons through the channel.Although the tissue-specific expressio...

Membrane proteins are key players in all living cells. To achieve a better understanding of membrane protein function, significant amounts of purified protein are required for functional and structural analyses. Overproduction of eukaryotic membrane proteins, in particular, is thus an essential yet non-trivial task. Hence, improved understanding of factors which direct a high production of eukaryotic membrane proteins is desirable. In this study we have compared the overproduction of all human aquaporins in the eukaryotic host Pichia pastoris. We report quantitated production levels of each homologue and the extent of their membrane localization. Our results show that the protein production levels vary substantially, even between highly homologous aquaporins. A correlation between the extents of membrane insertion with protein function also emerged, with a higher extent of membrane insertion for pure water transporters compared to aquaporin family members with other substrate specificity. Nevertheless, the nucleic acid sequence of the second codon appears to play an important role in overproduction. Constructs containing guanine at the first position of this codon (being part of the mammalian Kozak sequence) are generally produced at a higher level, which is confirmed for hAQP8. In addition, mimicking the yeast consensus sequence (ATGTCT) apparently has a negative influence on the production level, as shown for hAQP1. Moreover, by mutational analysis we show that the yield of hAQP4 can be heavily improved by directing the protein folding pathway as well as stabilizing the aquaporin tetramer.

Screening of protein variants requires specific detection methods to assay protein levels and stability in crude mixtures. Many strategies apply fluorescence-detection size-exclusion chromatography (FSEC) using green fluorescent protein (GFP) fusion proteins to qualitatively monitor expression, stability, and monodispersity. However, GFP fusion proteins have several important disadvantages; including false-positives, protein aggregation after proteolytic removal of GFP, and reductions in protein yields without the GFP fusion. Here we describe a FSEC screening strategy based on a fluorescent multivalent NTA probe that interacts with polyhistidine-tags on target proteins. This method overcomes the limitations of GFP fusion proteins, and can be used to rank protein production based on qualitative and quantitative parameters. Domain boundaries of the human G-protein coupled adenosine A2a receptor were readily identified from crude detergent-extracts of a library of construct variants transiently produced in suspension-adapted HEK293-6E cells. Well expressing clones of MraY, an important bacterial infection target, could be identified from a library of 24 orthologs. This probe provides a highly sensitive tool to detect target proteins to expression levels down to 0.02 mg/L in crude lysate, and requires minimal amounts of cell culture.

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