Ctf7p/Eco1p exhibits acetyltransferase activity–but does it matter?

In eukaryotic cells, faithful chromosome segregation depends upon the physical pairing, or cohesion, between sister chromatids. Budding yeast CTF7/ECO1 (herein termed CTF7) encodes an essential protein required to establish cohesion during S-phase and associates with DNA replication factors [1–10]. However, the molecular mechanism by which Ctf7p establishes cohesion remains unknown. In vitro characterization of Ctf7p as an acetyltransferase led to the model that this activity provides for Ctf7p's essential function [11]. However, in vivo Ctf7p substrates have yet to be documented, nor has an in vivo acetyltransferase activity been demonstrated even when Ctf7p is overexpressed [11] (A. Brands and R.V. Skibbens, unpublished data). In fact, the effects of acetylation-defective Ctf7p (ctf7 ack-) in yeast remain to be rigorously tested, leaving unanswered the critical questions of whether Ctf7p acetyltransferase activity is essential for cell viability and to what extent this activity is required for the establishment of cohesion. Here, we show that yeast strains harboring acetyltransferase-defective alleles [11] as the sole source of Ctf7p function exhibit robust growth and high fidelity chromosome transmission. We first used a plasmid loss assay to test whether ctf7 ack-alleles could support cell viability. CEN TRP1 plasmids containing wild-type CTF7 or ctf7 ack-alleles exhibiting abrogated or greatly diminished acetyltransferase activity in vitro [11] were transformed into an ade2;ade3 yeast assay strain in which the sole source of Ctf7p function was provided by a CEN-ADE3-CTF7 plasmid. After growth on medium selective for both plasmids, transformants were placed on rich non-selecting medium to allow for random plasmid loss. Cells transformed with CEN-TRP1-CTF7 exhibited CEN-ADE3-CTF7 plasmid loss — an event easily detected by white sectors in an otherwise red colony [12]. Cells transformed with vector alone produced solid red colonies, confirming that the CEN-CTF7-ADE3 plasmid was required in these cells. Importantly, three of the four ctf7 ack-alleles produced white-sectored colonies identical to cells transformed with wild-type CTF7 (Figure S1, Supplemental data). A fourth allele produced thin and infrequent white sectored colonies, but was competent to perform the essential function of Ctf7p. To test whether the resulting ctf7 ack-strains might exhibit conditional growth phenotypes, serial dilutions were spotted onto non-selective rich medium plates and incubated at 23°C, 30°C or 37°C. Three of the ctf7 ack-mutant strains exhibited wild-type growth at all temperatures tested (Figure S2, Supplemental data). Plasmid rescue and DNA sequencing confirmed that the ctf7 ack-alleles (tested for R222G/K223G) provided the sole source of Ctf7p function. In summary, these …