Molecular cloning and characterization of Aquaporin-Z: a water channel from E. coli

摘要

The aquaporin family of molecular water channels is\udwidely expressed throughout the plant and animal kingdoms.\udNo bacterial aquaporins are known; however, sequence-\udrelated bacterial genes have been identified that\udencode glycerol facilitators (glpF). By homology cloning,\uda novel aquaporin-related DNA (aqpZ) was identified\udthat contained no surface N-glycosylation consensus.\udThe aqpZ RNA was not identified in mammalian mRNA\udby Northern analysis and exhibited bacterial codon usage\udpreferences. Southern analysis failed to demonstrate\udaqpZ in mammalian genomic DNA, whereas a\udstrongly reactive DNA was present in chromosomal DNA\udfrom Escherichia coli and other bacterial species and\uddid not correspond to glpF. The aqpZ DNA isolated from\udE. coli contained a 693-base pair open reading frame\udencoding a polypeptide 28–38% identical to known aquaporins.\udWhen compared with other aquaporins, aqpZ\udencodes a 10-residue insert preceding exofacial loop C,\udtruncated NH2 and COOH termini, and no cysteines at\udknown mercury-sensitive sites. Expression of aqpZ\udcRNA conferred Xenopus oocytes with a 15-fold increase\udin osmotic water permeability, which was maximal after\ud5 days of expression, was not inhibited with HgCl2, exhibited\uda low activation energy (Ea 5 3.8 kcal/mol), and\udfailed to transport nonionic solutes such as urea and\udglycerol. In contrast, oocytes expressing glpF transported\udglycerol but exhibited limited osmotic water permeability.\udPhylogenetic comparison of aquaporins and\udhomologs revealed a large separation between aqpZ and\udglpF, consistent with an ancient gene divergence.