Analyses of the Effects of All Ubiquitin Point Mutants on Yeast Growth Rate

Abstract

The amino acid sequence of a protein governs its function. We used bulk competition and focused deep sequencing to investigate the effects of all ubiquitin point mutants on yeast growth rate. Many aspects of ubiquitin function have been carefully studied, which enabled interpretation of our growth analyses in light of a rich structural, biophysical and biochemical knowledge base. In one highly sensitive cluster on the surface of ubiquitin almost every amino acid substitution caused growth defects. In contrast, the opposite face tolerated virtually all possible substitutions. Surface locations between these two faces exhibited intermediate mutational tolerance. The sensitive face corresponds to the known interface for many binding partners. Across all surface positions, we observe a strong correlation between burial at structurally characterized interfaces and the number of amino acid substitutions compatible with robust growth. This result indicates that binding is a dominant determinant of ubiquitin function. In the solvent inaccessible core of ubiquitin all positions tolerated a limited number of substitutions, with hydrophobic amino acids especially interchangeable. Some mutations null for yeast growth were previously shown to populate folded conformations indicating that for these mutants subtle changes to conformation caused functional defects. The most sensitive region to mutation within the core was located near the C-terminus that is a focal binding site for many critical binding partners. These results indicate that core mutations may frequently cause functional defects through subtle disturbances to structure or dynamics.

Pivotal role for the ubiquitin Y59-E51 loop in lysine 48 polyubiquitination

“…Together, these results strongly suggest that the Ub Y59-E51 loop is the predominant determinant governing the production of cellular K48-linked polyubiquitin chains in mammals. Consistent with this, a recent high-throughput screen identified that point mutations to Ub Y59 resulted in an overall reduced fitness of yeast cultures (12). Although the importance of the Ub Y59-E51 loop extends to one other K48-specific E2, Ubc1 (SI Appendix, Fig.…”

Deep mutational scanning of an RRM domain of the Saccharomyces cerevisiae poly(A)-binding protein

“…The deep mutational scanning approach has been used to study the in vivo effect of all possible 171 single amino acid substitutions across a 9-amino acid-long stretch of the yeast Hsp90 (Hietpas et al 2011) and many of the possible 1425 single amino acid substitutions in the 75-amino acid-long ubiquitin sequence (Roscoe et al 2013). However, we found Deep mutational scanning of an RRM domain www.rnajournal.org 1547 that current yeast methods and sequencing technology allow an in vivo assessment of nearly two orders of magnitude more variants, which enables an analysis of variants with multiple mutations while still maintaining high coverage for variants with a single-point mutation.…”

A Statistical Guide to the Design of Deep Mutational Scanning Experiments

“…Mutants are constructed by transformation of preconstructed plasmid mutant libraries, each representing one of all total point mutations from the focal protein region; these then undergo bulk competition for a number of generations. Fitness is determined by assessing relative growth rates from the relative abundance of each mutant, which is obtained from deep sequence data for a number of time points.To date, EMPIRIC has been applied to yeast (Saccharomyces cerevisiae) to illuminate the DFE of all point mutations in Ubiquitin (Roscoe et al 2013) and Hsp90 (Hietpas et al 2011) across different environments, to quantify the amount and strength of epistatic interactions within a region of Hsp90 , and to assess a large intragenic fitness landscape in Hsp90. Recently, this approach has been extended to human influenza A virus to study the DFE in a region of the Neuraminidase protein containing a known drug-resistant locus.…”

“…To date, EMPIRIC has been applied to yeast (Saccharomyces cerevisiae) to illuminate the DFE of all point mutations in Ubiquitin (Roscoe et al 2013) and Hsp90 (Hietpas et al 2011) across different environments, to quantify the amount and strength of epistatic interactions within a region of Hsp90 , and to assess a large intragenic fitness landscape in Hsp90. Recently, this approach has been extended to human influenza A virus to study the DFE in a region of the Neuraminidase protein containing a known drug-resistant locus.…”