A major requirement for strain improvement involves stress tolerance and adaptability of cells to environmental stressors in industrial applications (22
). For example, some industrial processes involving industrial strains of Saccharomyces cerevisiae
, like brewing and some wine fermentations, take place at temperatures around 10 to 12°C, which is far below the optimal temperature for this organism (~28°C). Growth at low temperature reduces the production by S. cerevisiae
of higher alcohols and increases the amount of esters (10
). The low fermentation temperature also has a prominent effect on primary flavors, which are retained to a greater degree. However, under these conditions, an extended lag phase before the onset of vigorous fermentation activity is observed, which reduces the cost-effectiveness and efficiency of production. In wine making, this lag phase also increases the risk of halted or sluggish fermentation (20
). Hence, cold tolerance is an important biotechnological trait and there is an urgent need for strains able to ferment at low temperatures both quickly and in a reproducible way.
Like other stressors, cold influences the structural and functional properties of cellular components negatively, both physically and chemically (4
). Cold modifies enzyme kinetics (28
) and increases the molecular order of membrane lipids, i.e., rigidification (33
), affecting the membrane environment and thus the activity of membrane-associated enzymes and transporters. Key processes such as plasma membrane ATPase activity (53
), the higher proton motive force (39
), and the transport of various amino acids (60
) depend thus on temperature-instigated changes in membrane fluidity and become limiting factors for cell growth.
In support of this view, previous reports have shown that tryptophan uptake is impaired after a downward shift in temperature (1
) and that several cold-sensitive mutants are affected in tryptophan transport and biosynthesis (3
). It has also been suggested that the sensitivity of tryptophan permeases to changes in membrane fluidity could determine or influence the growth temperature profile of tryptophan auxotroph strains of S. cerevisiae
). Bearing this is mind, we hypothesized that enhanced membrane fluidity might rescue the cold-mediated growth inhibition of Trp−
yeast strains (48
). Indeed, production in S. cerevisiae
of sunflower desaturases (encoded by FAD2
genes) increased the content of dienoic fatty acids and fluidity of the yeast membrane; however, growth was diminished in the recombinant FAD2
strains at low temperatures (48
). Thus, membrane fluidity appears to be essential in determining cold tolerance in S. cerevisiae
, although its exact effect on amino acid uptake and growth is unclear.
The impact on yeast physiology of cold stress also depends on how low temperature affects the composition of membrane proteins. It has been reported that cold triggers the degradation of the tryptophan transporter Tat2p via the ubiquitination pathway (2
). Ubiquitination is a reversible posttranslational modification of cellular proteins, playing a central role in the regulation of protein degradation and trafficking (31
). In particular, Rsp5p-dependent ubiquitination of Tat2p has been observed in starved yeast cells (8
). The yeast ubiquitin ligase Rsp5p (a homolog of mammalian Nedd4 family proteins) controls most trafficking-related ubiquitination events at the plasma membrane and at other membranes (9
). In agreement with this, overproduction of Tat2p, or mutation of DOA4
, or UBP14
, encoding ubiquitin-specific proteases, confers increased cold growth to Trp−
yeast cells (1
). Altogether, these results suggest that Rsp5p may play a role in the degradation of Tat2p in response to low temperature, although no direct evidence of this function has been reported. Likewise, the putative adaptor proteins involved in this regulatory process are unknown, nor is it known whether cold may trigger the downregulation of other membrane proteins.
Here, we have performed a multicopy suppressor analysis of the cold sensitivity phenotype of a trp1 mutant strain of S. cerevisiae. Our hypothesis was that this screening could reveal, among others, genes governing plasma membrane properties. However, all the genes identified encode proteins directly or indirectly involved in ubiquitination machinery. We have exploited these effects to identify previously uncharacterized actors and genetic interactions that appear to be important in remodeling the membrane protein repertoire at low temperature. Finally, we demonstrate that engineering the ubiquitination machinery can potentially reverse growth inhibition associated with cold stress in industrial strains.