Molecular and Cellular Biology, September 2001, p. 5733-5741, Vol. 21, No. 17
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.17.5733-5741.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Department of Plant and Microbial Biology, University of California, Berkeley, California 94720,1 and Department of Biology, San Francisco State University, San Francisco, California 941322
Received 19 January 2001/Returned for modification 22 March 2001/Accepted 4 June 2001
Methylammonium and ammonium (MEP) permeases of Saccharomyces
cerevisiae belong to a ubiquitous family of cytoplasmic
membrane proteins that transport only ammonium
(NH4+ + NH3). Transport and
accumulation of the ammonium analog [14C]methylammonium,
a weak base, led to the proposal that members of this family were
capable of energy-dependent concentration of the ammonium ion,
NH4+. In bacteria, however, ATP-dependent
conversion of methylammonium to
-N-methylglutamine by
glutamine synthetase precludes its use in assessing concentrative
transport across the cytoplasmic membrane. We have confirmed that
methylammonium is not metabolized in the yeast S. cerevisiae and have shown that it is little metabolized in the
filamentous fungus Neurospora crassa. However, its
accumulation depends on the energy-dependent acidification of vacuoles.
A
vph1 mutant of S. cerevisiae and a
vma1 mutant, which lack vacuolar H+-ATPase activity, had large (fivefold or greater)
defects in the accumulation of methylammonium, with little accompanying
defect in the initial rate of transport. A vma-1 mutant of
N. crassa largely metabolized methylammonium to
methylglutamine. Thus, in fungi as in bacteria, subsequent
energy-dependent utilization of methylammonium precludes its use in
assessing active transport across the cytoplasmic membrane. The
requirement for a proton gradient to sequester the charged species
CH3NH3+ in acidic vacuoles provides
evidence that the substrate for MEP proteins is the uncharged species
CH3NH2. By inference, their natural substrate
is NH3, a gas. We postulate that MEP proteins facilitate diffusion of NH3 across the cytoplasmic membrane
and speculate that human Rhesus proteins, which lie in the same domain family as MEP proteins, facilitate diffusion of CO2.
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