Karyopherin-mediated nuclear import: Mechanistic insights revealed through the characterization of Kap121p and cargoes

The nuclear envelope divides eukaryotic cells into the nuclear and cytoplasmic compartments. In Saccharomyces cerevisiae there are at least 14 soluble nuclear transport receptors, termed karyopherins, which govern the continuous flow of proteins, nucleic acids and small molecules between these two compartments. Knowledge of the cargoes carried by each karyopherin and insight into the mechanisms of transport are fundamental to understanding constitutive and regulated transport, and elucidating how transport impacts normal cellular functions. Yeast Kap121p was studied as a model karyopherin to enhance our understanding of how transport machinery interfaces with its cargoes and how these interactions affect cellular physiology. New kap121 mutants were generated and proteomics identified 27 candidate Kap121p import cargoes. Two novel phenotypes associated with kap121 mutants were observed: Defects in mating and pseudohyphal/invasive growth. Ste12p, a transcription factor central to both of these cellular processes, was among the candidate Kap121p cargoes. Characterization of the Kap121p-Ste12p interaction demonstrated that Kap121p interacts directly with Ste12p and that its function is required for Ste12p import. Additionally, mutations within KAP121 specifically blocked Ste12p-induced transcription and the differentiation pathways it mediates. Together these data clearly demonstrate that seemingly pleiotropic phenotypes associated with kap mutants can be directly attributed to the mislocalization of a cargo imported by that beta-kap.; Nop1p, Dbp9p and Sof1p were also among the Kap121p-interacting proteins identified and were found to be bona fide Kap121p cargoes. Studies characterizing Nop1p import revealed a new class of Kap121p recognized NLS, distinguished by the abundance of arginine and glycine residues (termed rgNLSs). KAP104, a karyopherin that also recognizes rg-NLSs, was shown by several criteria to functionally overlap with Kap121p. Thus, these apparently unrelated transport pathways converge, creating a nuclear transport network that mediates the import of some common cargoes. Additionally, these studies revealed a novel piggy-back mechanism for cargo import, where Nop1p bridges the interaction between Sof1p and Kap121p. These data highlight the complex network of interactions between import karyopherins and their cargoes, and define additional levels of redundancy and flexibility built into nuclear transport pathways to ensure timely cargo delivery.