Role Of The TORC1-Sch9 Signaling Pathway In The Regulation Of Yeast-to-hypha Transition In The Yeast Yarrowia Lipolytica

Some fungal species can grow either in the ovoid yeast form or in the filamentous form.The conversion between these two forms is called dimorphic transition.Dimorphic transition in fungi is thought to be a foraging behavior in response to nutrient limitation.It is very important for cell survival when nutrients become scarce.It is also critical for some pathogenic fungi during the invasion to the hosts.Studying the regulatory mechanisms that control dimophic transition is of great scientific importance to the elucidation of how fungi adapt to the environment.Yarrowia lipolytica is an oleagineous yeast that can grow using alkanes,fatty acids and oils as the sole carbon source.Y.lipolytica is distantly related to Saccharomyces cerevisiae and Candida albicans.Like S.cerevisiae and C.albians,Y.lipolytica can also undergo dimorphic transition when induced by environmental factors such as nitrogen starvation,neutral pH or the presence of serum.We are interested in the dimorphic transition in Y.lipolytica and wish to understand its regulatory mechanism.S.cerevisiae and C.albicans are the two major yeast species dedicated to the study of fungal dimorphic transition.Since dimorphic transition can be induced by nutrient limitation,most of the previous studies are investigating the mechanisms by which cells sense nutrient limitation and turn on dimorphic transition.Under nutrient abundance,whether there is particular mechanism in the cell to repress dimorphic transition is not understood.The TORC1 protein complex is an important regulator of cell growth and metabolism in eukaryotes.It can coordinate nutrient sensing with cell proliferation.It has been reported that the TORC1-Sch9 signaling pathway can repress dimorphic transition under low O2 but high CO2.This discovery inspired us to explore the role of TORC1-Sch9 pathway in the regulation of dimorphic transition in Y.lipolytica.Glycerol and glucose are two favorite carbon sources for Y.lipolytica.We found that Y.lipolytica cells adopt an oval yeast-form shape when growing in culture medium with glycerol as the sole carbon source.In contrast,cells grown in culture medium with glucose as the sole carbon source are in the filamentous form.The cells are markedly elongated.We also observed that glycerol can repress filamentous growth induced by glucose whereas glycerol depletion induces filamentous development.These findings indicate that dimorphic transition in Y.lipolytica can be repressed by glycerol but not by glucose.Our results indicate that the repression of dimorphic transition by glycerol is mediated by the TORC1-Sch9 signaling pathway.When TORC1 activity is inhibited by rapamycin or caffeine or YlSCH9 gene is deleted,Y.lipolytica cells can grow in the filamentous form even though glycerol is abundant in the culture medium.These results demonstrate that TORC1-Sch9 signaling pathway represses dimorphic transition.Further analysis on YlSch9 revealed that the two phosphorylation sites T470 and T634 as well as the C-terminal region are important for YlSch9's function in regulating dimorphic transition.TORC1 and YlSch9 also regulate cell growth in Y.lipolytica.The regulation on dimorphic transition and cell growth are independent on each other.It was reported previo.usly that the TORC1-Sch9 pathway in C.albicans represses dimorphic transtion.However,the downstream target of TORC1-Sch9 pathway remains elusive.We investigated the function of TORC1-Sch9 pathway's potential downstream target—protein kinase YlRiml5.We observed that YlRiml5 positively regulates dimorphic transition as YIRIM15 deletion caused defect in filamentous growth.Because Ylsch9A mutant cells are filamentous whereas Ylsch9? Ylrim15? mutant cells are non-filamentous,YlRim15 may function as a downstream target of the TORC1-Sch9 sinaling pathway.We found that TORC1-Sch9 can inhibit the nuclear transloction of YlRim15.In Ylsch9 A mutant or TORC1-inhibited cells(treated by caffeine),GFP-YlRim15731-1604,an YlRim15 segment,displayed enrichment in the nucleus.Although full-length YlRim15 did not show enrichment in the nucleus in Ylsch9? cells,we observed that YlRiml5 ?NES1,which has one nucleus export signal NES1 removed,was enriched in the nucleus.This finding suggests that YlRiml5 can be transported out of the nucleus directed by NES motifs.This process prevents the enrichment of YlRim15 in the nucleus.We also observed that the Ylriml5?NES1 mutant can not rescue the filamentous growth defect of Ylrm15? mutant,suggesting that dynamic shuttling between the cytoplasm and the nucleus,rather than staying inside the nucleus,is critical for YlRim15's function in the control of dmorphic transition.We found that the transcription factor YlSko1 may be the other downstream target of TORC1-Sch9 pathway apart from YlRim15.YlSko1 promotes filamentous differentiation as its deletion markedly reduced filamentation.Our results show that YlSko1 functions downstream of YISch9 and it regulates dimorphic transition in parallel to YlRim15,We found that YlRiml5 does not control dimorphic transition via the two transcription factors YlMsn4 and YlGisl.YlRim15 may function via other transcription factors.We identified MHY1,a transcription factor-encoding gene,as a target gene regulated by YlRim15.We propose that the TORC1-Sch9 signaling pathway controls dimorphic transition through YlRim15,which in turn controls the transcription of target genes including MHYl.Our results show that the TORC1-Sch9 signaling pathway in Y.lipolytica represses dimorphic transition when glycerol is abundant.We identified YlRim15 and YlSko1 as two effectors that function downstream of TORC1-Sch9 in the control of dimorphic transition.These findings provide new insights in the understanding of regulation on dimorphic transition in fungi.