Tackling an essential problem in functional proteomics of Saccharomyces cerevisiae
© BioMed Central Ltd 2003
Published: 24 September 2003
Gene inactivation is the cornerstone of functional genetic analysis, but the analysis of essential genes requires conditional inactivation of the gene product. A new study has adapted a simple method for creating conditional alleles to allow large-scale analysis of essential genes in Saccharomyces cerevisiae and has identified a role in DNA replication for a newly identified protein complex.
Over thirty years ago, Hartwell and colleagues recognized that the identification and analysis of genes controlling the eukaryotic cell cycle represented a special challenge (and opportunity) because many, if not most, cell-cycle genes would be essential for viability . The isolation of conditional, temperature-sensitive (ts) mutant strains of yeast and subsequent phenotypic screening of these strains led to the identification of many cell-division-cycle (CDC) genes, setting the stage for tremendous progress in understanding the eukaryotic cell cycle . Today, the availability of complete genome sequences and the advent of proteomic methods promise similar advances in determining the functions of large numbers of uncharacterized genes. In one example, high-throughput gene deletion methods have been used to construct a collection of Saccharomyces cerevisiae mutant strains each containing a deletion in one non-essential gene . The study found that 18% of the roughly 6,200 genes in the S. cerevisiae genome were essential (because haploid strains with deletions in these genes could not be isolated), and therefore could not be analyzed further. Because most genes with a known role in chromosomal DNA replication are essential and a significant number of essential genes in yeast remain uncharacterized, it seems likely that this 18% includes novel DNA-replication genes and is ripe for analysis. But, analysis of these genes requires a method to allow conditional inactivation of each of the gene products. A recently developed method facilitates analysis of essential genes in S. cerevisiae and has revealed a function in DNA replication for a newly identified protein complex .
Conditional mutations, and most notably ts mutants, have been used with great success to analyze essential genes: certain amino-acid changes in a protein destabilize its structure at an elevated (or lowered) temperature . The potential for generating a useful ts mutation in any given gene is not equal, however, because some gene products are less prone to such destabilizing mutations than others; hence, some genes may elude identification in traditional (random) genetic screens for conditional alleles . Although in vitro mutagenesis can be used to generate ts mutations directly in individual genes of interest , these methods are not suited to the rapid analysis of large numbers of genes.
To analyze gene function in yeast on a large scale, methods enabling relatively simple genome modification with universal DNA cassettes that contain repressible promoters have been developed [6–8]. Although these methods are often successful at greatly reducing the levels of gene transcription and eventually protein, rapid and sufficient elimination of protein function remains a problem for many proteins. This is particularly true of cell-cycle studies, where slow gene inactivation can activate one or more of the cell-cycle checkpoints, which block the cell cycle if earlier steps are not completed adequately (reviewed in ), and can thereby mask the actual arrest point for a mutant, limiting the direct functional insights.
A potential solution to this problem would be a method that modified individual genes such that the resulting gene product is tagged for conditional elimination of its function. In principle, the most effective way of eliminating the function of any protein would be through complete proteolysis. Varshavsky and colleagues developed the thermolabile Degron, an ammo-terminal protein-degradation signal that is targeted for proteolysis specifically at 37°C through the ubiquitin-mediated 'N-end rule' pathway, which selects aberrant proteins for degradation [10, 11]. Fusion of the Degron coding sequence to the amino terminus of a heterologous gene of interest creates a temperature-inducible Degron (td) fusion protein that may be susceptible to temperature-dependent proteolysis . Despite individual successes, degradation of many td fusion proteins is often not sufficiently efficient to eliminate their function .
Diffley and colleagues significantly ameliorated this problem by regulating the expression of UBR1, which encodes a recognition factor for proteolysis by the N-end pathway . Overproduction of UBR1 from the galactose-inducible (GAL) promoter significantly expands the range of td proteins that are rapidly and effectively degraded. This updated td approach has several advantages over the other methods discussed above. First, a time-consuming and possibly unsuccessful search for a ts allele can be avoided. Second, the function of td proteins is typically normal at the permissive condition of 23°C (and even at 37°C when GAL-UBR1 is off), unlike ts proteins that are often partially defective under permissive conditions. And third, degradation of the td proteins, and thus elimination of their function, can be very rapid and effective at 37°C when GAL-UBR1 is induced. This method has been used to re-examine a number of essential replication factors individually, enabling detailed analysis of their functions [12, 13].
Focusing on Cdc102 and Cdc105, Labib's group  examined the effect on DNA synthesis of depleting each protein at different points during the cell cycle. The efficient degradation of Cdc102-td and Cdc105-td allow clear replication defects to be detected in cells arrested before and after the establishment of replication forks, strongly suggesting a role for these proteins in the establishment and progression of replication forks. These 'execution point' experiments are commonly used in cell-cycle studies to determine the requirement for an individual factor in different cell-cycle processes and to determine the order of action of different factors within a process [2, 17]. The success of experiments of this type relies strongly on efficient elimination of gene function, emphasizing the value of the td approach.
The main problem encountered with the use of td alleles remains the insufficient or ineffective proteolysis of a significant number of td fusion proteins. About 40% of the td strains in the Labib lab study  were viable, clearly indicating incomplete protein inactivation. Nevertheless, some of these, such as CDC101-td, remain informative, especially given the increasing numbers of potential interacting proteins identified by various large-scale experiments that may suggest additional candidates for analysis. In addition, the Degron cassette also introduces the hemagglutinin (HA)-epitope tag into the td protein, facilitating immediate characterization of its abundance and stability by western blot (Figure 1). The majority of td strains are therefore likely to be informative and their utility is limited mainly by the availability of appropriate assays for the many diverse biological functions represented in the mutant collection. Other potential problems, such as lethality (under any condition) due to replacement of the native gene promoter with the CUP1 promoter or to inactivity of the gene fusion itself, were relatively infrequent: only 6% of the td strains were inviable . The overall ease of strain construction and analysis makes the td allele the current method of choice for initial phenotypic analysis of essential gene function in S. cerevisiae.
In principle, the td approach should be applicable to all eukaryotes, as the N-end pathway is conserved, but efficient proteolysis in other eukaryotes has not yet been achieved [18, 19]. In addition, significant improvements in gene targeting will be required before this approach can be applied on a genome-wide scale in multicellular organisms. Until then, the functional characterization of the yeast proteome using td alleles will continue to reveal essential functions shared by all eukaryotes.
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