Msn5p is involved in formaldehyde resistance but not in oxidative stress response in the methylotrophic yeast Candida boidinii

Methylotrophic yeasts, which can utilize methanol as sole carbon and energy source, are exposed to two toxic metabolic intermediates, formaldehyde and hydrogen peroxide, during growth on methanol. Here we report that Msn5p, an importin-β family nuclear exporter, participated in the formaldehyde resistance mechanism but not in the hydrogen peroxide resistance mechanism in Candida boidinii. Disruption of the MSN5 gene in this yeast caused retardation of growth on formaldehyde-generating growth substrates such as methanol and methylamine, but the expression levels of the methanol-metabolizing enzymes did not fall. The Msn5p-depleted strain was sensitive to formaldehyde but not to hydrogen peroxide. Furthermore, a yellow fluorescent protein-tagged Msn5p was diffuse in the cytoplasm of C. boidinii when the cells were treated with high concentrations of formaldehyde or ethanol, but was predominantly associated with the nuclei following treatment with hydrogen peroxide.     

The carrier Msn5p/Kap142p promotes nuclear export of the hsp70 Ssa4p and relocates in response to stress

Cytoplasmic hsp70s like yeast Ssa4p shuttle between nucleus and cytoplasm under normal growth conditions but accumulate in nuclei upon stress. This nuclear accumulation is only transient, and Ssa4p relocates to the cytoplasm when cells recover. We show here that Ssa4p nuclear export is independent of Xpol/Crm1 and identify the importin-beta family member Msn5p/Kap142p as the exporter for Ssa4p. In growing cells and in vitro, Msn5p and Ssa4p generate genuine export complexes that require Ran/Gsp1p-GTP. Furthermore, nucleoporin Nup82p, which plays a role in Msn5p-mediated transport, is necessary for efficient export of Ssa4p. In living cells, stress not only regulates Ssa4p localization, but also controls the distribution of Msn5p. Msn5p is concentrated in nuclei of unstressed cells, but appears in the cytoplasm upon exposure to ethanol, heat, starvation or severe oxidative stress. In addition, growth on non-fermentable carbon sources relocates a portion of Msn5p to the cytoplasm and leads to a partial nuclear accumulation of Ssa4p. Taken together, growth and stress conditions that localize the transporter Msn5p to the cytoplasm also induce the nuclear accumulation of its cargo Ssa4p.     

A loss-of-function mutation in the PAS kinase Rim15p is related to defective quiescence entry and high fermentation rates of Saccharomyces cerevisiae sake yeast strains

Sake yeast cells have defective entry into the quiescent state, allowing them to sustain high fermentation rates. To reveal the underlying mechanism, we investigated the PAS kinase Rim15p, which orchestrates initiation of the quiescence program in Saccharomyces cerevisiae. We found that Rim15p is truncated at the carboxyl terminus in modern sake yeast strains as a result of a frameshift mutation. Introduction of this mutation or deletion of the full-length RIM15 gene in a laboratory strain led to a defective stress response, decreased synthesis of the storage carbohydrates trehalose and glycogen, and impaired G(1) arrest, which together closely resemble the characteristic phenotypes of sake yeast. Notably, expression of a functional RIM15 gene in a modern sake strain suppressed all of these phenotypes, demonstrating that dysfunction of Rim15p prevents sake yeast cells from entering quiescence. Moreover, loss of Rim15p or its downstream targets Igo1p and Igo2p remarkably improved the fermentation rate in a laboratory strain. This finding verified that Rim15p-mediated entry into quiescence plays pivotal roles in the inhibition of ethanol fermentation. Taken together, our results suggest that the loss-of-function mutation in the RIM15 gene may be the key genetic determinant of the increased ethanol production rates in modern sake yeast strains.