Functional expression and characterization of an archaeal aquaporin. AqpM from methanothermobacter marburgensis.
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Researchers have described aquaporin water channels from diverse eubacterial and eukaryotic species but not from the third division of life, Archaea. Methanothermobacter marburgensis is a methanogenic archaeon that thrives under anaerobic conditions at 65 °C. After transfer to hypertonic media,M. marburgensis sustained cytoplasmic shrinkage that could be prevented with HgCl2. We amplified aqpM by PCR from M. marburgensis DNA. Like known aquaporins, the open reading frame of aqpM encodes two tandem repeats each containing three membrane-spanning domains and a pore-forming loop with the signature motif Asn-Pro-Ala (NPA). Unlike other known homologs, the putative Hg2+-sensitive cysteine was found proximal to the first NPA motif in AqpM, rather than the second. Moreover, amino acids distinguishing water-selective homologs from glycerol-transporting homologs were not conserved in AqpM. A fusion protein, 10-His-AqpM, was expressed and purified from Escherichia coli. AqpM reconstituted into proteoliposomes was shown by stopped-flow light scattering assays to have elevated osmotic water permeability (P f = 57 μm·s−1 versus12 μm·s−1 of control liposomes) that was reversibly inhibited with HgCl2. Transient, initial glycerol permeability was also detected. AqpM remained functional after incubations at temperatures above 80 °C and formed SDS-stable tetramers. Our studies of archaeal AqpM demonstrate the ubiquity of aquaporins in nature and provide new insight into protein structure and transport selectivity. To withstand environmental and physiological stresses, organisms must be able to rapidly absorb and release water. Facilitated transport of water across cell membranes must be highly selective to prevent uncontrolled movement of other solutes, protons, and ions. Discovery of the aquaporins provided a molecular explanation to these processes (2). More than 200 aquaporins have now been identified, and their presence has been established in most forms of life (3). No aquaporin from Archaea has yet been characterized, although functional roles for a water channel protein have been predicted in these organisms (4). Two major protein family subsets are presently recognized, water-selective channels (aquaporins) and glycerol-transporting homologs with varying water permeabilities (aquaglyceroporins). The permeation selectivity of new members of the protein family may be predicted by a small number of conserved residues (5, 6). Several prokaryotic aquaporins and aquaglyceroporins are known. The bacterial water channel, AqpZ, was first identified in Escherichia coli (7, 8). Movement of water across the bacterial plasma membrane may be part of the osmoregulatory response by which microorganisms adjust cell turgor (9), although the regulation and physiological role of AqpZ are being reassessed (10). AqpZ is a highly stable tetramer with negligible permeability to glycerol. In contrast, the glycerol permeability of the glycerol facilitator (GlpF) fromE. coli has long been recognized (11). GlpF has relatively limited water permeability (12), and the tetrameric form has reduced stability in some detergents (13). Atomic resolution structures have been solved for GlpF (14) as well as human and bovine AQP11 (15-17). These have elucidated differential specificities and functional mechanisms of the two sequence-related proteins. Archaea and certain other microorganisms are able to withstand exceptional challenges in maintaining water balance as they thrive in extreme environments including saturated salt solutions, extreme pH, and temperatures up to 130 °C (18). We recently recognized the DNA sequence of AqpM, a candidate aquaporin or aquaglyceroporin in the genome of a methanogenic thermophilic archaeon,Methanothermobacter marburgensis 2 (,19). Here we investigate water permeability in living cells and report the purification, functional reconstitution, and characterization of AqpM.