Regulating protein function or protein level is often useful in order to investigate diverse biological processes. The fission yeast Schizosaccharomyces pombe is a popular model organism. It is genetically tractable and a wide variety of methods have been developed to facilitate molecular genetic manipulations in S. pombe.
It is usually more advantageous to regulate the activity of the target protein than the protein level, because this results in faster regulation of the protein's activity at wild type protein levels. The most commonly used approach to regulate the activity of the protein of interest is the isolation of conditional mutants, which have been vital tools in many areas of research. Indeed, one of the many advantages of fission yeast as a model system is that it is haploid, which makes it easier to isolate and work with conditional mutants. Most conditional mutants are temperature sensitive. However, not all genes can be mutated such that the corresponding protein becomes temperature sensitive. Furthermore, a temperature shift in itself might stress the cells. Temperature-sensitive proteins often have considerable residual activity at the restrictive temperature such that they rescue the temperature-sensitive mutant when overexpressed. Another common problem is that many temperature-sensitive proteins are not fully active at the permissive temperature. Therefore, temperature shifts of temperature-sensitive mutants are frequently far from the ideal "on" and "off" states that might be desired when regulating protein function. The reversibility of the inactivation varies greatly from mutant to mutant. Upon shift back to the permissive temperature, some temperature-sensitive proteins regain their activity, thus allowing block-and-release experiments. However, many other temperature sensitive proteins do not regain their activities after a period of temperature shift or are degraded at the restrictive temperature. Temperature-sensitive mutants have been particularly useful to explore the functions of essential proteins. However, it is difficult to identify temperature-sensitive mutants of non-essential genes, unless their function is known so that appropriate screens can be designed.
Regardless of the many advantages associated with the use of conditional mutants, they are not always available or applicable. A commonly used alternative is regulating the level of the protein of interest, either by regulating transcription or by regulating protein degradation (see below).
Numerous plasmids have been designed for regulated expression of genes , but there are no good tight and rapidly inducible promoters for use in fission yeast. The nmt1 (n o m essage in t hiamine) promoter was the first regulatable promoter to be described in fission yeast  and it remains the most commonly used one. This promoter is strong, but mutated versions with reduced strengths are available . The promoter is repressed by thiamine (vitamin B1). The main drawback with the nmt promoter is that induction of protein expression is rather slow and it takes several generations to achieve full activation, presumably because the cellular vitamin pools have to be depleted first. Furthermore, thiamine confers over 100-fold repression of nmt1-driven transcription, but the promoter is still somewhat leaky and many cloned genes are expressed to near wild-type levels even in the presence of thiamine, such that they can complement chromosomal mutations. Shut-off experiments, where expression of the protein of interest is turned off by the addition of thiamine, are particularly inefficient for stable proteins, since not only is the promoter leaky, but the protein of interest also has to be diluted out as the cells grow.
There are several other and less widely used regulatable promoters that to some extent can be used in fission yeast. Although they confer regulated expression, there are also severe drawbacks to their use, as detailed below. The tetracycline regulatable promoter is a derivative of the Cauliflower Mosaic Virus (CaMV) promoter, fused to a tetracycline binding site . The use of this promoter requires not only cloning the gene of interest behind the CaMV promoter but also manipulating the parent strain such that it expresses the Tet repressor. The fbp1 promoter is repressed by glucose but it can only be used in liquid cultures . The invertase promoter is also repressed by glucose and is activated by sucrose within an hour of medium shift. However, the glucose produced by invertase activity leads to repression of the promoter within a short time, so this promoter can only be used for short periods of expression . Since regulation of the latter two promoters requires changing the carbon source, their use implies dramatically changing the growth conditions during the course of the experiment.
Only recently has a uracil-regulateable promoter been described that allows rapid activation and inactivation of transcription . This system is expected to become a useful tool to regulate protein expression, but it should be noted that it might not always be sufficient to regulate transcription levels to achieve efficient regulation of protein levels.
The above regulatable systems all employ heterologous promoters. The expression levels from these promoters might or might not correspond to that from the native promoter of the gene of interest. The degron method, that circumvents this drawback, is based on regulated degradation of the target protein and has been used successfully in fission yeast [8–10]. However, it depends on a temperature shift to 37°C and the degron tag must be on the N-terminus of the target protein. Depending on the stability of the protein of interest, additional measures might also need to be taken to inactivate the protein. One improvement to the method in fission yeast was to combine the degron with an existing temperature sensitive mutation [8, 9]. Another strategy that was employed in budding yeast is overexpression of the ubiquitin ligase Ubr1 [10, 11]. This approach however cannot be used in fission yeast to improve degron-directed degradation 
In summary, despite having a selection of approaches to regulate protein levels, fission yeast researchers often find it difficult to achieve the desired expression level of their favourite proteins.
Here we describe the application of a system that is based on regulated protein function [12, 13] without the need for a temperature shift, as opposed to regulated transcription or protein degradation. We have tested the system on four proteins and were able to regulate the activity of two of them.