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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.

Evidence that Fungal MEP Proteins Mediate Diffusion of the Uncharged Species NH3 across the Cytoplasmic Membrane

Eric Soupene,1 Robert M. Ramirez,2 and Sydney Kustu1,*

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 gamma -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 Delta vph1 mutant of S. cerevisiae and a Delta 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.

* Corresponding author. Mailing address: Department of Plant and Microbial Biology, 111 Koshland Hall #3102, University of California, Berkeley, CA 94720-3102. Phone: (510) 643-9308. Fax: (510) 642-4995. E-mail: kustu{at}nature.berkeley.edu.

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.


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