Kar5p Is Required for Multiple Functions in Both Inner and Outer Nuclear Envelope Fusion in Saccharomyces cerevisiae

Abstract

During mating in the budding yeast Saccharomyces cerevisiae, two haploid nuclei fuse via two sequential membrane fusion steps. SNAREs (i.e., soluble N-ethylmaleimide–sensitive factor attachment protein receptors) and Prm3p mediate outer nuclear membrane fusion, but the inner membrane fusogen remains unknown. Kar5p is a highly conserved transmembrane protein that localizes adjacent to the spindle pole body (SPB), mediates nuclear envelope fusion, and recruits Prm3p adjacent to the SPB. To separate Kar5p’s functions, we tested localization, Prm3p recruitment, and nuclear fusion efficiency in various kar5 mutants. All domains and the conserved cysteine residues were essential for nuclear fusion. Several kar5 mutant proteins localized properly but did not mediate Prm3p recruitment; other kar5 mutant proteins localized and recruited Prm3p but were nevertheless defective for nuclear fusion, demonstrating additional functions beyond Prm3p recruitment. We identified one Kar5p domain required for SPB localization, which is dependent on the half-bridge protein Mps3p. Electron microscopy revealed a kar5 mutant that arrests with expanded nuclear envelope bridges, suggesting that Kar5p is required after outer nuclear envelope fusion. Finally, a split-GFP assay demonstrated that Kar5p localizes to both the inner and outer nuclear envelope. These insights suggest a mechanism by which Kar5p mediates inner nuclear membrane fusion.

C. elegans pronuclei fuse after fertilization through a novel membrane structure

“…It is likely that parental chromosome merging requires the activity of a membrane fusion machinery, the nature of which remains to be determined. An obvious candidate for this process is the protein Kar5/Brambleberry, which is involved in nuclear and karyomere fusion in yeast and zebrafish, respectively (Abrams et al, 2012; Rogers and Rose, 2014). However, we and others (Ning et al, 2013) have not been able to find a Kar5/Brambleberry homologue in C. elegans .…”

“…The only system in which merging of parental genomes is understood at a molecular level is nuclear fusion during budding yeast mating. In this system, the two nuclei fuse in a two-step process: first, the two outer nuclear membranes fuse, followed by fusion of the two inner ones, generating a single “fusion pore” (Melloy et al, 2007; Rogers and Rose, 2014).…”

Analysis of membrane proteins localizing to the inner nuclear envelope in living cells

“…These modified reporters can be introduced into strains generated in this study to test localization to other subcellular compartments to further refine our knowledge of protein localization and targeting at the single-cell level. Recent work from Rogers and Rose (2014) illustrates the utility of split-GFP in the detection of Kar5 at the INM and ONM during nuclear membrane fusion in yeast. In addition, split-GFP can be used to determine protein topology, as we showed for Mps3 and Heh2.…”

In-Depth High-Throughput Screening of Protein Engineering Libraries by Split-GFP Direct Crude Cell Extract Data Normalization

“…Regarding the use of our approach for the screening of yeast-expressed proteins exported via the secretory pathway, there is in principle no reason to think that the GFP11 tag could be incompatible with the yeast translocation system, as other small flexible tags have been successfully employed before. In fact, the GFP11 tag has been previously used for the detection of transmembrane proteins in plant, yeast, and animal cells (Hyun et al, 2015;Rogers and Rose, 2014). As the translocation mechanisms required for protein secretion and transmembrane protein expression are similar, it can be reasonably assumed that the GFP11 tagging would not constitute a problem for the heterologous expression and secretion of proteins in yeast.…”