Distinct Subcellular Localization Patterns Contribute to Functional Specificity of the Cln2 and Cln3 Cyclins of Saccharomyces cerevisiae

Abstract

The G₁ cyclins of budding yeast drive cell cycle initiation by different mechanisms, but the molecular basis of their specificity is unknown. Here we test the hypothesis that the functional specificity of G₁ cyclins is due to differential subcellular localization. As shown by indirect immunofluorescence and biochemical fractionation, Cln3p localization appears to be primarily nuclear, with the most obvious accumulation of Cln3p to the nuclei of large budded cells. In contrast, Cln2p localizes to the cytoplasm. We were able to shift localization patterns of truncated Cln3p by the addition of nuclear localization and nuclear export signals, and we found that nuclear localization drives a Cln3p-like functional profile, while cytoplasmic localization leads to a partial shift to a Cln2p-like functional profile. Therefore, forcing Cln3p into a Cln2p-like cytoplasmic localization pattern partially alters the functional specificity of Cln3p toward that of Cln2p. These results suggest that there are CLN-dependent cytoplasmic and nuclear events important for cell cycle initiation. This is the first indication of a cytoplasmic function for a cyclin-dependent kinase. The data presented here support the idea that cyclin function is regulated at the level of subcellular localization and that subcellular localization contributes to the functional specificity of Cln2p and Cln3p.

ACKNOWLEDGMENTS

We thank Mike Rout and David Lawrence for advice and helpful discussions in these studies. We thank Ray Deshaies for providing the anti-Cdc28p antibody, Mike Rout for providing the anti-Nop1p antibody, David Lawrence for providing peptide substrates, and Tony Gartner and Mike Tyers for providing plasmids. We also thank Mary Koszelak, Kristi Levine, Kimberly Huang, Maria Yuste Rojas, and David W. Miller for helpful discussions.

This work was supported by NIH grant GM47238 to F.R.C. M.E.M. was supported by an NRSA GM18782.