During ascospore formation in Saccharomyces cerevisiae, the secretory pathway is reorganized to create new intracellular compartments, termed prospore membranes. Prospore membranes engulf the nuclei produced by the meiotic divisions, giving rise to individual spores. The shape and growth of prospore membranes are constrained by cytoskeletal structures, such as septin proteins, that associate with the membranes. Green fluorescent protein (GFP) fusions to various proteins that associate with septins at the bud neck during vegetative growth as well as to proteins encoded by genes that are transcriptionally induced during sporulation were examined for their cellular localization during prospore membrane growth. We report localizations for over 100 different GFP fusions, including over 30 proteins localized to the prospore membrane compartment. In particular, the screen identified IRC10 as a new component of the leading-edge protein complex (LEP), a ring structure localized to the lip of the prospore membrane. Localization of Irc10 to the leading edge is dependent on SSP1, but not ADY3. Loss of IRC10 caused no obvious phenotype, but an ady3 irc10 mutant was completely defective in sporulation and displayed prospore membrane morphologies similar to those of an ssp1 strain. These results reveal the architecture of the LEP and provide insight into the evolution of this membrane-organizing complex.C omprehensive localization studies have provided a wealth of information about the functions of different Saccharomyces cerevisiae proteins (1, 2). To date, most studies have examined protein localization only during mitotic growth in rich medium. The localization of proteins that are expressed only under specific conditions has not been systematically examined. Moreover, constitutively expressed proteins can also be relocalized under different conditions. Many examples of such changes in distribution occur when yeast cells undergo sporulation (3-6).When diploid yeast cells are starved for nitrogen in the presence of a nonfermentable carbon source, they exit the mitotic cycle and enter the developmental program of meiosis and sporulation (7). Spores are created in an unusual cell division in which membranes are formed de novo in the cytosol and enclose each of the daughter nuclei produced by meiosis. These prospore membranes initially form on the cytoplasmic face of each of the four spindle pole bodies (SPBs) present in meiosis II. The membranes then expand beyond the SPBs to engulf the nuclei. As they do so, their shape is constrained by membrane-associated protein complexes.One of these membrane-associated complexes, the leadingedge protein complex (LEP), composed of the proteins Ssp1, Ady3, and Don1, forms a ring structure at the lip of the prospore membrane (8-10). The LEP is organized in a stratified fashion, with SSP1 being required for the localization of Ady3 and Don1 and ADY3 being required for the localization of Don1. The LEP helps to control the shape of the prospore membrane and is proposed to exert an outward for...