Fitness impact of N- versus C-terminal protein tagging. (a) Competition assay to compare the fitness of strains with N- or C-terminally tagged proteins. Pairs of strains with the same ORF tagged at the endogenous chromosomal locus are co-cultured. The percentage of each cell type in the population is followed with flow cytometry using strain-specific fluorescent markers expressed from the constitutive GPD promoter (mNG, green, or mCherry, magenta). The slope α of a linear fit to ln(mCherry⁺ cells/mNG⁺ cells) over time represents the relative fitness of the two strains (α > 0, competitive advantage of the ORF-Halo strain). (b) Genome-wide assessment of N- versus C-terminal protein tagging impact on fitness. Distribution of relative fitness for 5,247 ORFs assayed according to a. ORFs with α < −0.175 are defined as C-losers (C-terminal tagging impairs fitness, magenta area), α > 0.175 – N-losers (green area). Top, percentage of mCherry⁺ and mNG⁺ cells and linear fit to the ln(mCherry⁺/mNG⁺ cells) over time for three representative ORFs. (c) Percentage of ORFs differentially affected by N- versus C-terminal tagging as a function of a relative fitness threshold. Number of ORFs, essential (magenta) or non-essential (blue), differentially affected at two thresholds (dashed lines, abs(α) = 0.175 [5% fitness difference] and 0.611 [20%], Materials and methods) is indicated. (d) Frequency of relative fitness differences according to protein localization. Subcellular localization based on fluorescence microscopy imaging of 3,827 strains expressing C-terminally GFP-tagged proteins (Huh et al., 2003). (e) Proteins with differential localization of N- and C-terminally GFP tagged variants (Huh et al., 2003; Weill et al., 2018), stratified by gene essentiality and differential fitness according to b. N-loser, neutral, and C-loser ORFs defined at two relative fitness thresholds. Number of ORFs in each group is indicated.