Functional Expression and Characterization of an Archaeal Aquaporin

Abstract

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 HgCl 2 . 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 poreforming loop with the signature motif Asn-Pro-Ala (NPA). Unlike other known homologs, the putative Hg 2؉ -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 m⅐s ؊1 versus 12 m⅐s ؊1 of control liposomes) that was reversibly inhibited with HgCl 2 . 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) from E. coli has long been recognized (11). GlpF has relatively limited water perm...

Chlorella virus MT325 encodes water and potassium channels that interact synergistically

“…MIPs are present in all living organisms (7,8), where they form water channels either selective for water (aquaporins) or glycerolconducting channels, which also transport water, although to a lesser extent (aquaglyceroporins) (9). The viral MIP-like protein, named AQPV1 (aquaglyceroporin virus 1), was aligned and compared with the best-studied member of each group, aquaporin AQP1 (10, 11) from human and aquaglyceroporin GlpF (12) from Escherichia coli (Fig.…”

No facilitator required for membrane transport of hydrogen sulfide

“…An evolutionarily close relative of AqpM, the archeal aquaporin from a sulfidereducing bacteria Archaeoglobus fulgidus (AfAQP), shares 71% sequence identity with AqpM (compare SI Text for AfAQP sequence). In addition, the pore-forming amino acids in both of these aquaporins are identical except for replacement of cystein-79 of AqpM with alanine in AfAQP (16). The likelihood of AfAQP being an H 2 S pathway is high because aquaporin-mediated facilitation of water movement through the lipid matrix seems to be a superfluous luxury in hot springs.…”

Specific Aquaporins Facilitate the Diffusion of Hydrogen Peroxide across Membranes

“…All data presented are averages of at least 12 trace recordings. The rate constant of the decrease of scattered light intensity is proportional to the water permeability coefficient (25,26). Rate constants were calculated by fitting the curves using non-linear regression as described earlier (27).…”