Evidence for Separable Functions of Srp1p, the Yeast Homolog of Importin α (Karyopherin α): Role for Srp1p and Sts1p in Protein Degradation

Nucleocytoplasmic transport is a complex process mediated by interaction of transport cargoes with components of the nuclear pore complex along with other soluble factors (reviewed in references 9, 28, and 30). Import of most karyophilic proteins into the nucleus is energy dependent and mediated by short stretches of amino acids known as nuclear localization signals (NLSs). Proteins with classical NLSs, as exemplified by the simian virus 40 (SV40) large T antigen NLS and the nucleoplasmin NLS, are transported to the nucleus by a heterodimer composed of Srp1p (the Saccharomyces cerevisiae homolog of importin α [Kap60p]) and Kap95p (an S. cerevisiae homolog of importin β) (6, 10). Srp1p is the NLS receptor component that binds to the NLS itself, while Kap95p interacts with some components of the nuclear pore complex to facilitate movement of the Srp1p-Kap95p complex carrying a cargo protein from the cytoplasm to the nucleus (35, 36, 37). There are several other receptor proteins, which have amino acid sequence similarity to importin β (Kap95p) and are grouped in the importin β family. These proteins directly bind cargo proteins such as ribosomal proteins and carry out a protein import function independently of Srp1p (reviewed in references 9, 28, and 30). SRP1 was originally identified as a suppressor of certain temperature-sensitive (ts) mutations in RNA polymerase I (Pol I) in S. cerevisiae (54). Specific mutations in SRP1 were able to suppress ts mutations in the zinc-binding domains of RPA190 and RPA135, genes encoding the largest and second largest subunits of Pol I, respectively, but not other rpa190 and rpa135 ts mutations outside of this domain. These SRP1 suppressor mutations displayed no apparent phenotype on their own. Additional work has shown that different ts mutations in SRP1 cause different defects, which include protein import defects, nuclear segregation defects, altered microtubule morphology, and altered nucleolar morphology (24, 44, 55). Although the role of Srp1p in protein import is firmly established, it is not clear whether all of the diverse and allele-specific phenotypes displayed by these mutations are the consequences of defects in protein import. For example, while defects in NLS binding and NLS protein import were clearly demonstrated for the srp1-31 mutation (24, 44), no or only slight defects in NLS binding and protein import were observed for the srp1-49 mutation, which showed various other phenotypes (44, 55; unpublished data). Likewise, suppression of Pol I ts mutations by SRP1 mutations is difficult to explain on the basis of the Srp1p function as the NLS receptor (54, 55). For these reasons, we considered and tested the possibility that Srp1p might have an additional function(s) distinct from the NLS receptor function in protein import. We now report our discovery that two mutations, srp1-49 and srp1-31, show intragenic complementation, indicating that the function affected by srp1-49 is clearly different from the function affected by srp1-31, i.e., the NLS receptor function. To obtain clues to the functions affected by srp1-49, high-dosage suppressors of srp1-49 were isolated. Two suppressors identified, STS1 and RPN11, suppressed only srp1-49 and not srp1-31. STS1 was originally identified (22) as a dosage-dependent suppressor of a mutation in SEC23, a gene required for endoplasmic reticulum (ER)-Golgi protein transport (56). Independently, DBF8, which is identical to STS1, was also identified as a gene essential for nuclear segregation and division (12). RPN11 was recently identified as a component of the regulatory particle of the 26S proteasome (8), suggesting a link between SRP1 and the ubiquitin-proteasome system. Further experiments designed to examine such a link have led to the conclusion that Srp1p, together with Sts1p, carries out a function in the regulation of protein degradation by the ubiquitin-proteasome system and this function is separate from its established function in NLS-dependent protein import into the nucleus. We report these experiments and also discuss previous observations which support this conclusion.