Misregulation of 2μm Circle Copy Number in a SUMO Pathway Mutant

Abstract

Attachment of the ubiquitin-like protein SUMO to other proteins is an essential process in Saccharomyces cerevisiae. However, yeast mutants lacking the SUMO ligases Siz1 and Siz2/Nfi1 are viable, even though they show dramatically reduced levels of SUMO conjugation. This siz1Δ siz2Δ double mutant is cold sensitive and has an unusual phenotype in that it forms irregularly shaped colonies that contain sectors of wild-type-appearing cells as well as sectors of enlarged cells that are arrested in G₂/M. We have found that these phenotypes result from misregulation of the copy number of the endogenous yeast plasmid, the 2μm circle. siz1Δ siz2Δ mutants have up to 40-fold-higher levels of 2μm than do wild-type strains. Furthermore, 2μm is responsible for the siz1Δ siz2Δ mutant's obvious growth defects, as siz1Δ siz2Δ [cir⁰] strains, which lack 2μm, are no longer heterogeneous and show growth characteristics similar to those of the wild type. Possible mechanisms for SUMO's effect on 2μm are suggested by the finding that both Flp1 recombinase and Rep2, two of the four proteins encoded by 2μm, are covalently modified by SUMO. Our data suggest that SUMO attachment negatively regulates Flp1 levels, which may partially account for the increased 2μm copy number in the siz1Δ siz2Δ strain.

ACKNOWLEDGMENTS

We are very grateful to Virginia Zakian for calling nibbled to our attention. We thank Michael King's lab for use of their microscope, Eric Wickstrom's lab for use of their real-time PCR system, and Adam Dicker's lab for use of their LiCor Odyssey, which was generously loaned by LiCor. We also thank Greg Cesarone, Qing Ren, and Bill Davidson for help using equipment. We thank Gwen Bylebyl for the initial HA-His₈ tag construct and Aseem Gupta for technical assistance. We also thank Melanie Dobson for communicating results prior to publication, plasmids, and for comments on the manuscript and Marcus Shin for comments on the manuscript.

This work was supported by grants GM62268 from the National Institutes of Health and C0302 from the W. W. Smith Foundation.