We have shown that TIR1, TIR2, TIP1, and DAN1 are members of a larger group of homologous genes encoding mannoproteins involved in anaerobic adaptation. DAN2, DAN3, DAN4, TIR3, and TIR4 are all induced during hypoxia, but their expression is asynchronous, with expression of DAN2 and DAN3 being delayed for about 2 h after expression of the other DAN/TIR genes. We also found that the genes encoding the two major cell wall mannoproteins, CWP2 and CWP1, are down-regulated during anaerobic adaptation. In effect, it appears that oxygen deprivation results in extensive programmed remodeling of the cell wall, with Cwp1 and Cwp2 being replaced by the Dan/Tir proteins. Whether the latter proteins substitute functionally for Cwp1 and Cwp2 remains to be determined.
An analogous substitution process seems to occur when cells are subjected to cold shock, as TIR1
, and TIP1
are induced and CWP1
is down-regulated. In attempting to perceive a rationale for the replacement of one group of cell wall proteins by another during hypoxia and cold shock, one possibility is that the adaptation event is related to membrane fluidity. Cells subjected to these two seemingly unrelated environmental stresses both experience reduction in membrane fluidity—during hypoxia as a result of depletion of unsaturated fatty acids and during hypothermia as a result of membrane phase transition. The cell wall proteins transit the membrane during cell wall assembly; this process may be affected by membrane properties, and conceivably, alternate protein forms or variations in the mechanisms of secretion may accommodate differences in fluidity. Another possibility is that some of these proteins play a role in the transport of sterols, as suggested by the fact that the Mox4 regulator, which controls DAN/TIR
gene expression, also controls expression of factors involved in this process (5
). Clearly, the special function of the anaerobic cell wall proteins will be more obvious when the function of cell wall proteins and the mechanisms of cell wall assembly are better understood.
We observed that the critical induction temperature both for expression of TIP1
and down-regulation of CWP1
was 16 to 18°C. Hence, induction and down-regulation are half-maximal at a temperature in the range within which phase transition might be expected to occur (10
). Another observation suggesting that membrane fluidity is a factor in hypoxic adaptation was that unsaturated fatty acids repress hypoxic and cold shock-induced expression of TIP1
. Whether changes in membrane fluidity actually signal low-temperature induction of any of several genes showing this response remains to be determined. Clearly, not all cold shock-induced genes are activated by the same factors. We showed here that cold shock induction of TIR1
requires Mox4, whereas induction of OLE1
was not affected by the mox4
Δ allele (data not shown). Hence, if there is a common signal pathway it must diverge at the level of transcriptional regulators.
We noted a correspondence of homology and expression patterns, suggesting that there might be a functional basis for the difference in the patterns of regulation of these genes: (i) TIP1 and the TIR genes are clustered in a homology tree and are less stringently repressed by oxygen than the DAN genes; four TIR genes—TIP1, TIR1, TIR2, and TIR4—are induced by cold shock; (ii) the four DAN genes are also clustered and are stringently regulated and not induced by cold shock; and (iii) the two CWP genes, weakly homologous to the DAN/TIR genes, are quite similar and are induced by oxygen.
In general, the function of mannoproteins is not well understood, though there is evidence that Cwp1 and Cwp2 help maintain cell wall integrity, and a fatty acid esterase activity has been tentatively attributed to Tip1 (14
). As a first step in deducing the role of the Dan/Tir proteins, we tested the effect of deleting TIR1
, or TIR4
and found that each is essential for anaerobic growth. This observation was at variance with an earlier report for TIR1
), possibly because of strain differences or differences in the degree of hypoxia achieved in the growth chamber. Anaerobically grown tir
Δ cells were unbudded, presumably becoming arrested in G1
after onset of anaerobiosis. The clear growth phenotype of these mutants will facilitate structure-function analysis of the Tir proteins, including the role of the PAU domain.
We have reported elsewhere that a network of regulators is responsible for the coordinate regulation of the DAN/TIR
genes. This includes anaerobic induction by the Mox 4 transcriptional activator and aerobic repression by the Mox1 and Mox2 repressors. Some of the DAN/TIR
genes are also repressed during aerobic growth by Mot3, a repressor which is induced by heme and repressed in anaerobic cells by Hap1 (Sertil et al., submitted), in parallel with the Rox1 repressor (6
). Surprisingly, mutations affecting Mot3 and Mox4 also affected expression of CWP2
but in a manner opposite to their effect on DAN/TIR
expression, helping to account for induction of CWP2
in aerobic cells. We have concluded that Mot3, which is known to function as an activator or repressor of different genes through the same binding sites (13
), is an important activator of CWP2
, presumably through the three Mot3 sites in the promoter. It is interesting that the versatility of Mot3 allows it to mediate both positive and negative regulation by heme. Conversely, Mox4, the activator of the DAN/TIR
genes, plays a significant role in repressing CWP2
, though the mechanistic basis of this reciprocal effect is unknown. We also observed that the Tup1-Ssn6 repressor complex contributes to repression of anaerobic CWP2
expression, possibly by virtue of its role in repression of MOT3
(Sertil et al., submitted). CWP2
was earlier observed to be regulated during the cell cycle, as would be expected for a gene product associated with bud formation. Interestingly, analysis of genes regulated during the cell cycle revealed that expression of MOT3
is also cyclical, suggesting that fluctuations in Mot3 may account for cell cycle regulation of CWP2
Regulation of CWP1 is still not well understood, except for a descriptive list of signals affecting its expression, either positively (induction during the cell cycle or when cell wall synthesis is disrupted) or negatively, as shown here during hypoxic or hypothermal stress. Although CWP1 appears to be regulated by oxygen in parallel with CWP2, mot3 and mox4 mutations do not affect its expression, indicating that it is controlled by a different regulatory pathway. However, we did observe constitutive expression of CWP1 in a tup1Δ ssn6Δ strain during hypoxia, indicating that expression in that state is normally blocked by a repressor associated with Tup1-Ssn6. It is worth noting that even though TIP1 is regulated in parallel with the TIR genes, it is also controlled by a different pathway, showing no dependence on Mox4 or Mox1, Mox2, or Mot3 for repression by oxygen or induction by cold shock (data not shown). Work in this area has demonstrated that several distinct but interconnected mechanisms are deployed in yeast to achieve essentially the same effect, i.e., regulation by oxygen, targeting genes in several regulons.