Atg41 and Icy1 have overlapping functions. (A) Amino acid sequence alignment of Atg41 and Icy1. Sites of identity are bold and underlined, sites of similarity are bold. (B) GFP-expressing wild-type (WT) cells were mixed in equal number with either atg41Δ or icy1Δ cells and grown in either zinc-replete (ZR) or zinc-deficient (ZD) medium. Subsequently, the percentage of mutant cells remaining in the cultures after 15 generations of growth was measured by FACS. The fitness ratio for the atg41Δ mutant calculated from these data were 0.049, and the fitness ratio for the icy1Δ mutant was 0.983. (C) WT, icy1Δ, atg41Δ, and icy1Δ atg41Δ cells were grown in ZR or ZD media. (D) WT and atg41Δ cells were transformed with either empty vector or pICY1-OE, and grown in ZD medium. For all panels, cultures were assayed in triplicate and error bars represent 1 SD. The asterisks denote P < 0.05 by Students t-test.