Characterizing new ER and nuclear fusion pathways in Saccharomyces cerevisiae

Abstract

Lipid bilayer membrane fusion is universally essential for life, and most membrane fusion pathways are conserved across eukaryotes. However, with the exception of vesicle trafficking (mediated by SNARE proteins), most of these pathways are not well characterized. To better understand membrane fusion pathways, we first studied nuclear envelope fusion during mating in the budding yeast S. cerevisiae. Previous data suggested that the fusogen that directly catalyzes nuclear fusion might be essential for viability, and thus difficult to discover using classical genetic screens. Therefore, we screened mutants of fusogens from other pathways for nuclear fusion defects. We discovered that outer nuclear envelope fusion is mediated by a subset of SNAREs in a novel combination distinct from normal vesicle trafficking. Additionally, we discovered that homotypic ER-ER fusion, a process required for a normal, reticulated ER structure, occurs early during yeast mating and is necessary for the normal mating process. We further determined that there are two ER-ER fusion pathways: the canonical Sey1p-dependent pathway, and a second pathway mediated by SNAREs.

Pursuing these results, we characterized the SNARE-mediated homotypic ER fusion pathway during normal mitotic growth. We discovered that SNARE-mediated homotypic ER fusion additionally requires the Dsl1 complex, but not the COPI coat or any other complexes or pathways involved in normal vesicle trafficking. Therefore, perturbed vesicle trafficking does not result in apparent homotypic ER fusion defects, validating this new ER-ER fusion pathway and implying vesicle-independent functions for the Dsl1 complex.

Finally, because nuclear fusion during yeast mating requires the sequential fusion of both an outer and inner nuclear membrane (INM), we characterized the primary INM fusogen candidate, Kar5p. Kar5p is a highly conserved transmembrane protein that localizes to the nuclear fusion zone and is required for nuclear fusion. We performed a structure-function characterization and demonstrated that Kar5p localizes to the nuclear fusion zone in an Mps3p-dependent manner, recruits Prm3p, and performs at least one additional function after outer membrane fusion, including possibly physically coupling the outer and inner nuclear membranes, and directly catalyzing INM fusion. These data have informed new models of nuclear fusion.