The yeast 2-micron plasmid Rep2 protein has Rep1-independent partitioning function

Abstract

Equal partitioning of the multi-copy 2-micron plasmid of the budding yeast Saccharomyces cerevisiae requires association of the plasmid Rep1 and Rep2 proteins with the plasmid STB partitioning locus. Determining how the Rep proteins contribute has been complicated by interactions between the components. Here, each Rep protein was expressed fused to the DNA-binding domain of the bacterial repressor protein LexA in yeast harboring a replication-competent plasmid that had LexA-binding sites but lacked STB. Plasmid transmission to daughter cells was increased only by Rep2 fusion expression. Neither Rep1 nor a functional RSC2 complex (a chromatin remodeler required for 2-micron plasmid partitioning) were needed for the improvement. Deletion analysis showed the carboxy-terminal 65 residues of Rep2 were required and sufficient for this Rep1-independent inheritance. Mutation of a conserved basic motif in this domain impaired Rep1-independent and Rep protein/STB-dependent plasmid partitioning. Our findings suggest Rep2, which requires Rep1 and the RSC2 complex for functional association with STB, directly participates in 2-micron plasmid partitioning by linking the plasmid to a host component that is efficiently partitioned during cell division. Further investigation is needed to reveal the host factor targeted by Rep2 that contributes to the survival of these plasmids in their budding yeast hosts.

Figure 1.

Tethering Rep2 to an ARS plasmid lacking STB promotes plasmid inheritance that is not dependent on Rep1 or affected by absence of Rsc2. (A, B, D) Yeast cells lacking native 2μm plasmid were co-transformed with two plasmids: an ARS/CEN HIS3-tagged plasmid that would express either the DNA binding domain of LexA (LexA_BD) or the indicated protein fused to LexA_BD, and a TRP1-tagged ARS plasmid either containing (pTRP1/ARS/lexAop8) or lacking (pTRP1/ARS) an array of 8 LexA-binding sites. The co-transformed yeast cells were cultured overnight in medium selective for the presence of both plasmids. (C), as in (A), except yeast also contained a LEU2-tagged ARS/CEN plasmid that expressed an untagged version of the indicated 2μm plasmid protein under the control of a galactose-inducible promoter (GALp) or that did not encode a protein (–). The transformants were cultured overnight in medium selective for the presence of the plasmids and containing galactose as the carbon source. (A, C, D) The percentage of Trp⁺ cells (an indication of inheritance of the TRP1-marked plasmid) was determined by a plating assay. (D) As in (A) except the yeast lacked the RSC2 gene. Results represent the average (±s.d.) from assaying five independent co-transformants for each combination of plasmids for (A and D) and six for (C). Asterisks indicate significance of increased inheritance of the pTRP1/ARS/lexAop8 plasmid relative to the cells expressing only LexA_BD as determined by a two-tailed Student's t-test (*P< 0.05, **P< 0.01, ***P< 0.001, ^****P< 0.0001). (B) Total protein was extracted from the yeast transformants containing the pTRP1/ARS/lexAop8 plasmid used for the plating assay in (A) and analyzed by western blotting with antibodies specific for LexA (top) and a yeast host protein Pgk1 (bottom). Species with the mobility expected for the LexA fusion protein being expressed are indicated with an arrowhead. An open circle denotes a non-LexA host protein detected by the antibody.

Figure 2.

The C-terminal 65 residues of tethered Rep2 are required and sufficient for promoting plasmid inheritance in the absence of Rep1 and this activity is lost when a basic motif in this domain is mutated. Yeast lacking native 2μm plasmid were co-transformed with two plasmids: an ARS/CEN HIS3-tagged plasmid that would express either LexA_BD or LexA_BD fused to the indicated version of Rep2, either full-length (1–296), or truncated (1–231, or 58–296, or 232–296), and either wild type (WT), or mutant (D22N or L185A, L186A (AA)), or with four arginine-to-alanine substitutions in the C-terminal basic motif (4RA). The co-transformed yeast were cultured overnight in medium selective for the presence of both plasmids. (A) The percentage of Trp⁺ cells (an indication of inheritance of the TRP1-marked plasmid) was determined by a plating assay. Results represent the average (±s.d.) from assaying six independent co-transformants for each combination of plasmids. Asterisks indicate significance of increased inheritance of the pTRP1/ARS/lexAop8 plasmid relative to the cells expressing only LexA_BD as determined by a Student's two-tailed t-test (^****P< 0.0001). (B) Total protein was extracted from the co-transformed yeast cultures in (A) and analyzed by western blotting with antibodies specific for LexA (top) and a yeast host protein Pgk1 (bottom). Species with the mobility expected for the LexA_BD fusion protein being expressed are indicated with an arrowhead. An open circle denotes a non-LexA host protein detected by the antibody.

Figure 3.

Deletion of the C-terminal 65 residues of Rep2 or alteration of a basic motif in this domain impairs 2-micron plasmid inheritance. Yeast cells lacking native 2μm plasmid and with a deletion of the genomic ADE2 gene were transformed with ADE2-tagged amplification-defective (flp⁻) versions of a 2μm-based plasmid in which the REP2 gene was wild type (WT), or deleted (–), or had been replaced with one that encoded Rep2 residues 1–231 fused to the SV40 viral nuclear localization signal (+NLS) or full-length Rep2 with four arginine-to-alanine substitutions in the C-terminal domain basic motif (4RA). Transformed yeast cells were cultured in medium lacking adenine. The percentage of Ade⁺ cells (an indication of inheritance of the ADE2-marked 2μm plasmid) was determined by a plating assay. Results represent the average (±s.d.) from assaying four independent transformants for each. Data for the plasmid lacking REP2 are from a previous study (84). Asterisks indicate significance of differences as determined by a two-tailed Student's t-test (**P< 0.01, ^*****P< 0.000001).