Functional expression and characterization of an archaeal aquaporin. AqpM from Methanothermobacter marburgensis

David Kozono, Xiaodong Ding, Ikuko Iwasaki, Xianying Meng, Yoichi Kamagata, Peter Agre, Yoshichika Kitagawa

Research output: Contribution to journalArticle

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 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 poreforming 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 Pf = 57 μm·s-1 versus 12 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.

Original languageEnglish (US)
Pages (from-to)10649-10656
Number of pages8
JournalJournal of Biological Chemistry
Volume278
Issue number12
DOIs
StatePublished - Mar 21 2003

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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