Timely Closure of the Prospore Membrane Requires SPS1 and SPO77 in Saccharomyces cerevisiae

Abstract

During sporulation in Saccharomyces cerevisiae, a double lipid bilayer called the prospore membrane is formed de novo, growing around each meiotic nucleus and ultimately closing to create four new cells within the mother cell. Here we show that SPS1, which encodes a kinase belonging to the germinal center kinase III family, is involved in prospore membrane development and is required for prospore membrane closure. We find that SPS1 genetically interacts with SPO77 and see that loss of either gene disrupts prospore membrane closure in a similar fashion. Specifically, cells lacking SPS1 and SPO77 produce hyperelongated prospore membranes from which the leading edge protein complex is not removed from the prospore membrane in a timely fashion. The SPS1/ SPO77 pathway is required for the proper phosphorylation and stability of Ssp1, a member of the leading edge protein complex that is removed and degraded when the prospore membrane closes. Genetic dissection of prospore membrane closure finds SPS1 and SPO77 act in parallel to a previously described pathway of prospore membrane closure that involves AMA1, an activator of the meiotic anaphase promoting complex.KEYWORDS prospore membrane; meiotic exit; anaphase promoting complex; sporulation; cytokinesis; germinal center kinase B IOLOGICAL membranes provide a barrier for inhibiting the flow of materials between a cell and its surroundings and for compartmentalizing the contents of the various organelles within a cell. The size and shape of membranes are central to their functions. Membranes are essential for many fundamental cellular processes including ion balance, energy generation, and secretion. In cell division, membrane dynamics are particularly important, where they act to segregate the contents of the cellular progeny.The process of cell division differs among cells. Most commonly, animal cells divide through a mechanism requiring the assembly of an actin-based contractile ring at the division site (reviewed in Green et al. 2012). This actomyosin ring contracts and matures, ultimately leading to scission of the membrane necks mediated by the ESCRTIII (endosomal sorting complex required for transport III) complex. However, other variations of cytokinesis occur: cellularization during early Drosophila embryogenesis, which requires the growth of membranes around the nuclei before the contractile event (Lee and Harris 2014) and cell division in plants, which requires the secretion of vesicles to the division plane to form the phragmoplast, which will eventually separate the two daughter cells (reviewed in Jürgens 2005). Actin is not always involved in cytokinesis; although prokaryotic cells have actin-like filaments (reviewed in Carballido-López 2006), these filaments are not used for cytokinesis (Pollard and Wu 2010). Similarly, cytokinesis in Trypanosoma brucei (a bikont eukaryote) does not require actin but seems to utilize microtubules (Wheeler et al. 2013). In the budding yeast Saccharomyces cerevisiae, an actin-based contractile ring plays a role in ...

Efficient Sporulation of <em>Saccharomyces cerevisiae</em> in a 96 Multiwell Format

“…The sporulation efficiencies obtained using a glass bead provides a low-cost solution that creates enough aeration for high-throughput screening in a 96 multiwell format 16 . Unfortunately, the addition of a stir bar or a glass bead did not achieve the very high sporulation efficiencies (typically ~80% or greater) seen when sporulating in flasks, and thus subtle sporulation phenotypes may be difficult to detect when screening in a 96 well format.…”

“…We find that for this method, aeration is critical for synchronous and efficient sporulation, and have devised techniques to ensure sufficient sporulation a small-volume multiwell format. Sporulating in a 96 multiwell plate format allows for cells to be screened using high-throughput techniques and reagents optimized for a multiwell plate format, such screening for high copy suppressors using a tiled library [14][15][16] .…”

A non-canonical Hippo pathway regulates spindle disassembly and cytokinesis during meiosis inSaccharomyces cerevisiae

“…Samples were prepared as above and run at 80 V at 4°C 200 before being transferred and imaged, as above. Assaying prospore membrane closure, formation, and number 209 Cells were assayed for prospore membrane closure and formation as previously described 210 (Paulissen et al 2016). For prospore membrane closure and formation, only cells in anaphase II 211 (as determined by Htb2-mCherry) were counted.…”

“…254 cdc15 and spo77 were among the mutants identified in this screen that exhibited 255 decreased histone phosphorylation similar to sps1∆ ( Figure 1A). SPO77 was isolated as a high 256 copy suppressor of a hypomorphic allele of sps1 and acts with SPS1 in regulating timely 257 prospore membrane closure (Paulissen et al 2016). Because a link between SPS1 and CDC15 258 was not previously reported, we focused our studies on CDC15.…”

“…A 55 protein complex known as the Leading Edge Protein complex is at the growing edge of the 56 prospore membrane and includes Ssp1, Ady3, Irc10, and Don1 (Knop and Prospore membrane closure is the cytokinetic event in meiosis, and involves the removal 59 of the Leading Edge Protein complex (Maier et al 2007). Proper timing of prospore membrane 60 closure requires SPS1, which encodes a STE20-family GCKIII kinase; cells lacking SPS1 61 produce hyperelongated prospore membranes that close later than those in wild-type cells 62 (Slubowski et al 2014;Paulissen et al 2016). Prospore membrane closure must be properly 63 coordinated with other meiosis II events, such as spindle disassembly.…”