Pad1 : A novel subunit of the 26S proteasome in fission yeast
Mutations in the fission yeast genes mts5-1 and mbc]-1 were isolated in a screen for Schizosaccharomyces pombe (S.pombe) mutants that are both resistant to the microtubule destabilising drug methybenzylcarbamylate (MBCR) and temperature sensitive (t.s.) for growth. This screen has so far been specific for mutations in genes encoding subunits of the 26S proteasome (Gordon et al., 1993). This study shows that these strains contain mutations in the pad1 and crm] genes respectively. Crm] and pad] have previously been shown to be positive and negative regulators respectively of the AP-1 transcription factor Papi (Toda et al., 1992, Shimanuki et al., 1995), the S. pombe homologue of the mammalian AP-1 transcription factors fos and fun, which is involved in the transcription of multidrug resistance genes (Shimanuki et al., 1995). The mts5-1 (pad]-]) strain has a metaphase arrest phenotype and an increased level of high molecular weight ubiquitinated proteins when incubated at the restrictive temperature. This is identical to the mts2-1 (Gordon et al., 1993) and mts3-1 (Gordon et al., 1996) mutants isolated in the same screen and which have been shown to encode subunits of the 26S proteasome. This study reclassifies pad]' as a subunit of the 26S proteasome, 'and data is provided which shows genetic interactions between Padi and three other subunits of the 26S proteasome, Mts3 (Gordon et al., 1996), Mts4 (Wilkinson et al., 1997) and Pus (C. Wilkinson pers. comm.). A putative function for the 19S cap subunit Padi as an isopeptidase is also investigated. Crml has been implicated in MDR through Papi, since Papi is responsible for the transcription of genes involved in resistance to a wide variety of drugs. 26S proteasome mutants are also shown to be resistant to the same range of drugs as the mbcl-] (cnn]-I) mutant, but to a lower level, and that papli\ cells are sensitive to MBC (MBCS). 26S proteasome mutants are shown to have elevated levels of Papi when incubated at the permissive temperature indicating that this protein is not being degraded as efficiently as in wild type cells. A c.s. cnn] mutant has been shown to over-express a non-essential 25KDa protein that has been shown to be a downstream target of Papi (Adachi and Yanagida, 1989). This protein is also shown to be over expressed in S.pombe proteasome mutants and cnn]-1. paplA 26S proteasome double mutants are t.s. and MBCS. This is consistent with the 26S proteasome being involved in the degradation of Papi and hence involved in pleiotropic multi-drug resistance.