The General Amino Acid Permease FfGap1 of Fusarium fujikuroi Is Sorted to the Vacuole in

Abstract Therice pathogenicfungusFusariumfujikuroiiswellknown fortheproductionofabroadspectrum ofsecondarymetabolites (SMs) suchasgibberellicacids(GAs),mycotoxinsandpigments.Thebiosynthesisofmost oftheseSMs strictly depends onnitrogen availabilityandoftheactivityofpermeasesofnitrogen sources, e.g.theammonium andaminoacidpermeases.Oneofthe threeammonium permeases, MepB,wasrecentlyshowntoactnotonlyasatransporter but alsoasanitrogensensor affecting the productionofnitrogen-repressedSMs.Herewe describe theidentificationofageneral aminoacidpermease,FfGap1, amongthe99 putative aminoacidpermeases(AAPs) inthegenomeofF. fujikuroi.FfGap1 isable tofullyrestore growth oftheyeastgap1Δmutantonseveral aminoacidsin-cludingcitrullineandtryptophane.InS.cerevisiae,Gap1activityisregulatedbyshuttlingbe-tween theplasma membrane(nitrogenlimitingconditions)andthevacuole(nitrogensufficiency),whichwealsoshowfor FfGap1. Inyeast,the Npr1 serine/threoninekinasesta-bilizes the Gap1position atthe plasmamembrane.Here, weidentified andcharacterizedthreeNPR1-homologous genes, encodingthe putative protein kinasesFfNpr1-1,FfNpr1-2andFfNpr1-3 with significantsimilaritytoyeastNpr1.Complementationofthe yeastnpr1ΔmutantwitheachofthethreeF.fujikuroiNPR1homologues,resultedinpartialrestora-tionofammonium,arginineandproline uptake byFfNPR1-1whilenoneofthe threekinasesaffectgrowthon different nitrogensources andnitrogen-dependentsorting ofFfGap1 inF.fujikuroi. However, exchangeofthe putative ubiquitin-target lysine9(K9A)and15(K15A) residuesofFfGap1resultedinextendedlocalization totheplasma membraneandincreased proteinstability independentlyofnitrogen availability. Thesedata suggest asimi-larregulationofFfGap1by nitrogen-dependent ubiquitination,but differencesregarding theroleofFusariumNpr1 homologuescomparedtoyeast.