Regulation of PAU genes in Saccharomyces cerevisiae

How are PAU genes triggered under “stressed” conditions?

PAU Genes: Largest multi-gene family in yeast

Figure 1. PAU genes were first discovered in the wine fermentation process

PAU genes are the largest multigene family present  in yeast, with 24 members.  Nineteen copies are located in subtelomeric regions and five copies are found in internal regions, this is shown in Figure 2.  They are found on all of the sixteen yeast chromosomes and their function is not completely known (Luo, Z. , & van Vuuren, H., 2009). PAU genes are known to bring about the synthesis of the so-called seripauperines, a set of almost identical serine-poor proteins of unknown function (Goffeau, A., 1996). PAU genes were first observed to be expressed during alcohol fermentation.  Fermentation conditions include high glucose, low pH, low nitrogen availability and strong anaerobiosis (Luo, Z. , & van Vuuren, H., 2009).  This evidence along with the following experiments suggests that PAU genes are expressed during stress conditions and plays a role in the stress response of Saccharomyces cerevisiae.

Figure 2. Chromosomal location of PAU genes in S. cerevisiae strain S288C. The number in parentheses indicates the distance (in kb) of the PAU gene from the telomere end. Subtelomeric regions were defined by a distance of less than 20 kb from each telomere end (Wyrick et al., 1999). AA, amino acid; vertical lines indicate the relative location of PAU genes on the chromosomes; the filled circle represents the centromere.

PAU genes are regulated by anaerobiosis:

With further research by Martinez, Barre, and Blondin, it was found that PAU genes were not expressed when cells were grown on YNB-ethanol or YNB-glucose, YNB being a yeast nitrogen base without ammonium sulphate and amino acids.  To determine which of the conditions during fermentation plays the primary role in PAU gene expression, ϐ-galactosidase activity was measured in PAU-lacZ fusions under various conditions.  LacZ fusions are a way of studying regulation of gene expression.  All of the fusions showed low activity when grown in aerobic conditions and a dramatic increase in activity when grown in anaerobic conditions (Rachidi, N. , Martinez, M. , Barre, P. , & Blondin, B., 2000).  This is represented in Figure 3 shown below.

Figure 3. Northern Blot assay showing low activity when grown in aerobic conditions and increased activity when grown in anaerobic conditions.

Different PAU genes show different abilities to be induced by anaerobiosis: Related to chromosomal location

However, different gene fusions showed different levels of activation compared to others. PAU3-lacZ and PAU4-lacZ had much lower concentrations of ϐ-galactosidase activity than PAU5-lacZ.    This shows that different PAU genes have different abilities to be induced by anaerobiosis.  The location of the gene on the chromosome is proposed to have an effect on the ability to be induced by anaerobiosis.  PAU5 has an internal localization on chromosome VI, whereas PAU3 and PAU4 have subtelometric locations on chromosomes III and XII. Therefore this research suggests that subtelometric genes are more weakly inducible than an interstitial genes (Rachidi, N. , Martinez, M. , Barre, P. , & Blondin, B., 2000).  The results are shown graphically below in Figure 4.

Figure 4. The behavior of PAU5-lacZ fusion differed from that of the PAU3-lacZ and PAU4-lacZ fusions and expressed a ϐ-galactosidase activity 10 to 25-fold higher than the other two fusions throughout fermentation.

Repression by oxygen is mediated by an unknown, haem-dependant pathway:

As stated above, PAU genes are negatively regulated by oxygen.  This repression is mediated by an unknown haem-dependent pathway.  Haem is thought to be an oxygen sensor because its biosynthesis is linked to the availability of oxygen.  To determine if haem was involved in PAU gene regulation, it was added to anaerobic cultures.  The addition of haem to anaerobic cultures triggered an eight-fold reduction in ϐ-galactosidase activity.  The impact of haem on PAU mRNA abundance was also examined.  It was found that PAU mRNA were much less abundant after the addition of haem.  These results are shown in the Figure 5 below.  With this data, it was concluded that haem represses PAU genes and may mediate their repression under aerobic conditions (Rachidi, N. , Martinez, M. , Barre, P. , & Blondin, B., 2000).

Figure 5. a. To determine if haem was involved in PAU gene regulation. Addition of haem to anaerobic cultures triggered an eight-fold reduction in B-galactoside activity. b. Determine the impact of haem on PAU mRNA abundance. PAU mRNA were much less abundant after the addition of haem.

PAU repression by oxygen is independent of Rox1p but it involves Tup1p:

Lastly, PAU repression by oxygen was found to be independent of Rox1p.  However, it involves a general repressor, Tup1p.  ϐ-galactosidase activity driven by the PAU5-lacZ fusion was studied in the RZ53-6 rox1D strain, rox1Δ, and its wild-type form RZ53-6, and in the tup1D strain RTY418, tup1Δ, and its wild-type form RTY235.  The wild-type and the rox1Δ strains showed high ϐ-galactosidase activity under anaerobic conditions. Following that pattern, under aerobic conditions, expression of ϐ-galactosidase was weak in both rox1Δ and wild-type strains. This shows that PAU repression by oxygen is not relieved by the rox1 mutation.  The tup1Δ strain showed strong expression under both aerobic and anaerobic conditions.  This shows that Tup1p is involved in the repression of PAU genes by oxygen and that anaerobiosis does not lead to the full derepression of the PAU5 gene in this particular strain.  These results are shown in Figure 6 below (Rachidi, N. , Martinez, M. , Barre, P. , & Blondin, B., 2000).

Figure 6. ϐ-galactosidase activity driven by the PAU5-lacZ fusion was studied in the RZ53-6 rox1D strain, rox1Δ, and its wild-type form RZ53-6, and in the tup1D strain RTY418, tup1Δ, and its wild-type form RTY235.

Relationship of PAU proteins and their relatives

PAU proteins share homology with the N-terminal region of Tir and Dan proteins.  A phylogenetic was constructed to show these relationships (Figure 7).  This figure suggests that there are three clusters of PAU genes that encode proteins of 120, 122, 123/124 amino acids.  The PAU gene family is more closely related to the DAN family than to the TIR family.  From this data, it was suggested that DAN2 and DAN3 be renamed as PAU23 and PAU 24, respectively.

Figure 7. Phylogenetic relationships of PAU, DAN and TIR genes. The phylogenetic tree (neighbour-joining method) was produced with MEGA 3.1. The bootstrap test of phylogenies was performed in 1000 replicates. The cut-off value was set at 85.

What we know so far about PAU genes:

PAU expression was first detected during alcoholic fermentation.  Using Northern blot assays, it was determined that these genes were regulated specifically by anaerobiosis. However, it was found that genes have different abilities to be induced by anaerobiosis likely due to their location on the chromosome.  PAU genes are negatively regulated by oxygen through a haem-dependent pathway.  This pathway differs from others in that it does not involve Rox1p anaerobic repressor but requires Tup1p, a general repressor instead (Rachidi, N. , Martinez, M. , Barre, P. , & Blondin, B., 2000).

References:

Goffeau, A. (1996). Life with 6000 Genes.Science274(5287), 546.

Luo, Z. , & van Vuuren, H. (2009). Functional Analyses of PAU Genes in Saccharomyces Cerevisiae. Microbiology-sgm155(185601374), 4036-4049.

Rachidi, N. , Martinez, M. , Barre, P. , & Blondin, B. (2000). Saccharomyces Cerevisiae PAU Genes Are Induced by Anaerobiosis. Molecular Microbiology,35(6), 1421-1430.

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