University of Louisville
Further Characterization of the Ammonium Transporters of Smut Fungi
Institution
University of Louisville
Faculty Advisor/ Mentor
Michael Perlin
Abstract
Fungal ammonium transporters (known variously as Amts, Meps, or Umps) are members of a family of proteins that is conserved from bacteria to humans. Indeed, some members of this family have been found to be functionally equivalent across distant taxa. Interestingly, fungal Amts include proteins that not only transport ammonium, but also sense its availability. They somehow signal low abundance to pathways that trigger a switch from yeast-like to filamentous growth, a switch associated with pathogenesis by some fungi. We have continued to characterize the structure and function of Amts from the plant pathogenic smut fungi, Ustilago maydis and Microbotryum violaceum. We have cloned the mepC gene, a M. violaceum homologue, whose product is predicted to have 46% similarity with that of MepA (M. violaecuem), 53 % similarity with Ump1 and 40% similarity with Ump2 (U. maydis) and 41% similarity with Mep1 (S. cerevisiae). Ongoing experiments with this gene will examine its function in the yeast, S. cerevisiae, and in its smut cousin, U. maydis. In analogous experiments, we have introduced the MEP2 gene into an ump2- mutant and shown it to complement the filamentation defect of this mutant. Our major goal is to determine the proteins that interact directly with Umps in U. maydis to provide greater understanding of how such proteins “sense” ammonium availability and trigger the dimorphic switch. Towards this end, we have epitope-tagged the Ump2 protein and shown that the tagged protein functions in U. maydis. We have also used a yeast two-hybrid approach to identify possible interacting proteins. So far, three candidates of interest are a glutamate dehydrogenase, a membrane-bound acetyltransferase and a protein associated with trehalose metabolism that, in S. cerevisiae, also stimulates the switch to filamentous growth. The next step will be to confirm the interaction of these putative proteins with Ump2 by examining their co-localization in U. maydis cells.
Further Characterization of the Ammonium Transporters of Smut Fungi
Fungal ammonium transporters (known variously as Amts, Meps, or Umps) are members of a family of proteins that is conserved from bacteria to humans. Indeed, some members of this family have been found to be functionally equivalent across distant taxa. Interestingly, fungal Amts include proteins that not only transport ammonium, but also sense its availability. They somehow signal low abundance to pathways that trigger a switch from yeast-like to filamentous growth, a switch associated with pathogenesis by some fungi. We have continued to characterize the structure and function of Amts from the plant pathogenic smut fungi, Ustilago maydis and Microbotryum violaceum. We have cloned the mepC gene, a M. violaceum homologue, whose product is predicted to have 46% similarity with that of MepA (M. violaecuem), 53 % similarity with Ump1 and 40% similarity with Ump2 (U. maydis) and 41% similarity with Mep1 (S. cerevisiae). Ongoing experiments with this gene will examine its function in the yeast, S. cerevisiae, and in its smut cousin, U. maydis. In analogous experiments, we have introduced the MEP2 gene into an ump2- mutant and shown it to complement the filamentation defect of this mutant. Our major goal is to determine the proteins that interact directly with Umps in U. maydis to provide greater understanding of how such proteins “sense” ammonium availability and trigger the dimorphic switch. Towards this end, we have epitope-tagged the Ump2 protein and shown that the tagged protein functions in U. maydis. We have also used a yeast two-hybrid approach to identify possible interacting proteins. So far, three candidates of interest are a glutamate dehydrogenase, a membrane-bound acetyltransferase and a protein associated with trehalose metabolism that, in S. cerevisiae, also stimulates the switch to filamentous growth. The next step will be to confirm the interaction of these putative proteins with Ump2 by examining their co-localization in U. maydis cells.