The yeast Mid1 protein is an integral membrane protein required for the viability of differentiated cells and Ca2+ influx induced by mating pheromone. Our previous study has identified a loss-of-function mutation, F356S. The F356S mutant is completely unable to maintain viability, but still has Ca2+ accumulation activity near the wild-type level. Here we further examined in detail the F356S mutation to unravel the function of Phe(356). After exposure to the pheromone, the F356S mutant was not fully rescued by high extracellular Ca2+, like the mid1 null mutant, suggesting that Phe(356) and Mid1 itself are also required for viability maintenance mechanism that does not involve Ca2+ signalling. Substitutions of hydrophilic amino acids for Phe(356) caused lethality and low Ca2+ accumulation, but those of hydrophobic amino acids did not. Substitutions of small amino acids for Phe(356) caused a significantly reduced viability, but did not affect Ca2+ accumulation. We suggest that the hydrophobicity of the Phe(356) residue is important for both viability maintenance and Ca2+ uptake, and that its size for viability maintenance. (C) 2003 Elsevier Inc. All rights reserved.