Misregulation of 2 microm 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 siz1Delta siz2Delta 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 G(2)/M. We have found that these phenotypes result from misregulation of the copy number of the endogenous yeast plasmid, the 2 microm circle. siz1Delta siz2Delta mutants have up to 40-fold-higher levels of 2 microm than do wild-type strains. Furthermore, 2 microm is responsible for the siz1Delta siz2Delta mutant's obvious growth defects, as siz1Delta siz2Delta [cir(0)] strains, which lack 2 microm, are no longer heterogeneous and show growth characteristics similar to those of the wild type. Possible mechanisms for SUMO's effect on 2 microm are suggested by the finding that both Flp1 recombinase and Rep2, two of the four proteins encoded by 2 microm, are covalently modified by SUMO. Our data suggest that SUMO attachment negatively regulates Flp1 levels, which may partially account for the increased 2 microm copy number in the siz1Delta siz2Delta strain.

FIG. 1.

siz1Δ siz2Δ strains form irregularly shaped colonies containing enlarged cells. Wild-type (left) or siz1Δ siz2Δ (right) cells were streaked on YPD plates and incubated at 30°C for 1 day (bottom) or 3 days (top). Top panels show visible colonies, while images in bottom panels were taken with a tetrad dissecting microscope (see Materials and Methods).

FIG. 2.

siz1Δ siz2Δ strains contain elevated levels of the 2μm circle. (A) Genomic DNA was isolated from cultures of the indicated strains grown in YPD at 30°C. Equal amounts of DNA were digested with PstI and analyzed by Southern blotting. The four siz1Δ siz2Δ lanes represent four different isolates of the same strain (EJY326). Numbers under the lanes represent the relative amounts of 2μm DNA in each of these DNA samples as determined by qPCR (see Materials and Methods). Levels were normalized relative to that of the wild type. (B) Single large round colonies of wt (JD52) or siz1Δ siz2Δ (EJY326) strains that had been grown on YPD plates at 36°C were split and restreaked on three different YPD plates and incubated at 36, 30, or 20°C for 2 days. Genomic DNA was isolated from the resulting cells and analyzed by qPCR (see Materials and Methods). Each of the grouped sets of results represents cells from the same original colony grown at each of the three temperatures. Error bars reflect the difference between replicate PCRs using the same DNA sample.

FIG. 3.

Many phenotypes of the siz1Δ siz2Δ strain are suppressed by elimination of 2μm. (A) Cells of the indicated genotypes were streaked on a YPD plate and incubated at 20°C for 4 days. (B) Microscopy of wt (top), siz1Δ siz2Δ [cir⁺] (middle), or siz1Δ siz2Δ [cir⁰] (bottom) cells grown at 30°C using differential interference contrast (DIC) Nomarski optics (left) or DAPI staining (right). Bar, 5 μm. (C) Whole-cell lysates from cultures of the indicated genotypes were analyzed by SDS-PAGE and immunoblotting with an Ab against Rad9. Bands corresponding to Rad9 are indicated.

FIG. 4.

Isolates of many SUMO pathway mutants are [cir⁰]. Previously isolated SUMO pathway mutants were analyzed for the presence of 2μm DNA by colony PCR (see Materials and Methods), followed by agarose gel electrophoresis and ethidium bromide staining. The arrow indicates the band that is diagnostic for the presence of 2μm.

FIG. 5.

Flp1 and Rep2 are modified by SUMO. (A) Lysates from cells containing versions of 2μm in which different 2μm-encoded proteins were tagged with the HA epitope and a His₈ tag were subjected to Ni-NTA affinity chromatography (see Materials and Methods). Eluates were analyzed by SDS-PAGE and immunoblotting with Abs against HA (left) or Smt3 (right). Tagged proteins are indicated over the lanes. Bands corresponding to unmodified Rep1, Flp1, Rep2, and Raf1 are indicated. A band that cross-reacted with the anti-HA antibody is indicated by an asterisk. (B) Strains of the indicated genotypes containing 2μm with Flp1-HA-His₈ (left panels) or Rep2-HA-His₈ (right panels) were analyzed as in panel A using antibodies against HA (bottom panels) or Smt3 (top panels). Arrowheads indicate the unmodified proteins (there were two major bands of Rep2), and lines indicate SUMO-modified forms. Asterisks indicate bands that cross-reacted with the antibodies. Numbers under the lanes indicate the relative quantities of the unmodified Flp1 or Rep2 band, the monosumoylated band, and the amount of 2μm DNA in the same culture from which protein bands were quantified (see Materials and Methods). Quantities were normalized relative to the wild type. For Rep2, the first two sumoylated bands, which likely represent monosumoylation of the two different forms of Rep2, were pooled for protein quantification.

FIG. 6.

Lys³⁷⁵ of Flp1 is a major sumoylation site, and mutating it increases the amount of Flp1 protein. (A) Yeast strains of the indicated genotypes contained either native 2μm or 2μm with wild-type Flp1-HA-His₈ or Flp1-K375R-HA-His₈, as indicated. Analysis was as described in the legend to Fig. 5 with immunoblotting with anti-Smt3 (top) or anti-HA (bottom). Designations are as described in the legend to Fig. 5, except that sumoylated bands are designated with open circles placed on top of the image. Numbers under the lanes indicate the quantity of unsumoylated Flp1 relative to that in the strain with wild-type tagged Flp1. (B) Genomic DNA was isolated from cells of the indicated genotypes containing the indicated version of 2μm and analyzed by qPCR (see Materials and Methods). HH, HA-His₈ tag. Quantities were normalized to the value in wt cells containing unmodified 2μm. Error bars represent variations between DNA preparations from three separate isolates of each strain. (C) Microscopy of colonies from wild-type strains containing HA-His₈ versions of wild-type Flp1 (top) or K375R-Flp1 (bottom) that were grown for 2 days on a YPD plate at 20°C.