Genes that reduce fitness when under- but not over-expressed are enriched amongst protein complexes
Most essential functions of the eukaryotic cell are performed by multi-subunit protein complexes. As previously shown [8
], genes with essential functions are enriched amongst the subunits of multi-protein complexes (Figure ). This is also true for haploinsufficient genes (i.e. genes that reduce fitness when their dosage is reduced by half in heterozygotes [9
]) and for genes that cause slow growth when they are deleted [10
] (Figure ). Thus inhibiting the expression of a subunit of a protein complex is very likely to disrupt the function of that complex. However genes that slow growth when they are over-expressed [11
] (referred to here as genes with over-expression phenotypes) are not enriched amongst the subunits of protein complexes (Figure ). This lack of enrichment could reflect the fact that many protein complexes are not essential for normal growth and therefore perturbing their function will not result in a visible phenotype. However, we find that genes that reduce fitness when they are over-expressed are also not enriched amongst protein complexes that perform essential functions (Table ), nor are they enriched amongst the subunits of protein complexes that are essential when deleted (Table ). Thus in general over-expressing a subunit of an essential protein complex does not normally disturb its function.
Figure 1 Genes with under- but not over-expression phenotypes are enriched amongst protein complexes. Essential genes, genes required for normal growth in rich media and haploinsufficient genes are all enriched amongst the subunits of protein complexes. In contrast (more ...)
Protein complexes with essential functions are not enriched for subunits with over-expression phenotypes.
Genes with under- but not over-expression phenotypes cluster into individual protein complexes
Even if over-expressing a subunit of a protein complex does not in general disrupt the overall activity of the entire complex, it is still possible that a subset of protein complexes may be particularly sensitive to the over-expression of their subunits. To test this we investigated the distribution of genes with under-or over-expression phenotypes amongst complexes. For each phenotype we divided the protein complexes into ten evenly spaced bins according to the fraction of subunits associated with the phenotype. We then compared this distribution of phenotypes to that seen when the subunits are randomized amongst complexes.
As shown in Figure , genes with under-expression phenotypes (essential genes, haploinsufficient genes and genes required for normal growth) cluster into particular protein complexes. For example, 44 complexes have >90% essential subunits compared to 13 expected by chance, and for all phenotypes arising from decreased gene expression there are many more complexes with no genes having that phenotype than expected by chance. In contrast, for genes that reduce fitness when they are over-expressed, only two bins contain more complexes than expected by chance – one complex has 80–90% of tested subunits with an over-expression phenotype (compared to 0.01 expected, p = 0.006) and 5 complexes have >90% of tested subunits with an over-expression phenotype (1.54 expected, p = 0.02). Thus only a few complexes (~3/183) contain more subunits that are toxic when over-expressed than expected by chance. For the vast majority of complexes the distribution of genes with over-expression phenotypes is not different to that expected by chance.
Figure 2 Genes with under- but not over-expression phenotypes cluster into individual protein complexes. Genes with essential functions (A), genes required for normal growth in rich media (B), and haploinsufficient genes (C) are arranged amongst protein complexes (more ...)
To further confirm this conclusion we asked whether any individual protein complexes contain more subunits with over-expression phenotypes than expected by chance. To do this we randomised the assignment of subunits to protein complexes and for each complex counted the number of times it had the same or more subunits with an over-expression phenotype than seen with the real data. There are 9 complexes with more genes with over-expression phenotypes than in 5% of randomisations, but none of these are significantly enriched for over-expression phenotypes after adjusting for multiple hypothesis testing (see Supplementary table 1 in Additional file 1
, Benjamini-Hochberg false discovery rate, FDR = 5%). In contrast, there are 41 complexes with more essential genes than are seen in 5% of randomisations, and 17 of these complexes are still significantly enriched after adjusting for multiple hypothesis testing (see Supplementary table 2 in Additional file 1
, FDR = 5%). Indeed the complex most enriched for genes with over-expression phenotypes is the nucleosome complex, and here the over-expression phenotype may be more related to the disruption of the precise temporal regulation of histone expression during the cell cycle [12
] rather than disruption of protein complex formation per se
. Indeed there is an overall enrichment for genes with over-expression phenotypes amongst cell cycle regulated genes (p = 0.037, Fisher's exact test).
Thus we conclude that for protein complexes performing essential functions, inhibiting the expression of any subunit of a protein complex is likely to reduce the overall activity of that complex. In contrast, over-expressing any individual subunit of a protein complex does not normally inhibit the overall activity of that protein complex. This conclusion most likely applies to the vast majority of protein complexes in a eukaryotic cell.
Neither core nor peripheral subunits of protein complexes are enriched for genes with over-expression phenotypes
Previously it has been suggested that subunits that form the structural core of a protein complex might be particularly sensitive to alterations in expression level [13
]. Therefore we tested whether subunits with under- or over-expression phenotypes are enriched amongst the core or peripheral/isoform-specific subunits of protein complexes. In a genome-wide study of protein complexes identified by tandem affinity purification, Gavin et al.
identified a total of 491 complexes and classified their subunits as "core" – those present in most complex isoforms, "attachment" – those present only in some isoforms, and "modules" – two or more attachment proteins that tended to occur together in different complexes [3
]. As shown in Figure , there is no difference between the percentage of genes with over-expression phenotypes in cores, modules, or attachments when compared with yeast genes in general. In contrast, subunits with essential or haploinsufficient phenotypes are significantly enriched among all three types of subunit (p < 0.0001, Fisher's exact test). The same result is seen when only considering genes that fall exclusively within each classification, except that haploinsufficient genes are only enriched amongst attachments (Figure ).
Figure 3 Genes sensitive to a reduction in expression level, but not to over-expression are enriched amongst both the core and peripheral subunits of protein complexes. Percentages of genes with essential, overexpression or haploinsufficient phenotypes among different (more ...)
We conclude that complexes are often sensitive to reduction of a subunit from any part of the complex, and that isoform-specific subunits are particularly sensitive to a partial reduction in the expression of a subunit. These isoform-specific subunits are likely to be regulatory subunits (i.e. limiting the overall activity of a complex) and so may be particularly sensitive to a reduction in expression. In contrast there is no evidence that complexes are sensitive to the over-expression of any particular structural subclass of subunit. Our findings also do not support the previous prediction that the core subunits of protein complexes will be particularly sensitive to over-expression [13