Tagging of genes by chromosomal integration of PCR amplified cassettes is a widely used and fast method to label proteins in vivo in the yeast

Intracellular budding is a developmentally regulated type of cell division common to many fungi and protists. In Saccaromyces cerevisiae, intracellular budding requires the de novo assembly of membranes, the prospore membranes (PSMs) and occurs during spore formation in meiosis. Ssp1p is a sporulation-specific protein that has previously been shown to localize to secretory vesicles and to recruit the leading edge protein coat (LEP coat) proteins to the opening of the PSM. Here, we show that Ssp1p is a multidomain protein with distinct domains important for PI(4,5)P 2 binding, binding to secretory vesicles and inhibition of vesicle fusion, interaction with LEP coat components and that it is subject to sumoylation and degradation. We found non-essential roles for Ssp1p on the level of vesicle transport and an essential function of Ssp1p to regulate the opening of the PSM. Together, our results indicate that Ssp1p has a domain architecture that resembles to some extent the septin class of proteins, and that the regulated removal of Ssp1p from the PSM is the major step underlying cytokinesis in yeast sporulation.

During sporulation in Saccharomyces cerevisiae, the four daughter cells (spores) are formed inside the boundaries of the mother cell. Here, we investigated the dynamics of spore assembly and the actin cytoskeleton during this process, as well as the requirements for filamentous actin during the different steps of spore formation. We found no evidence for a polarized actin cytoskeleton during sporulation. Instead, a highly dynamic network of nonpolarized actin cables is present underneath the plasma membrane of the mother cell. We found that a fraction of prospore membrane (PSM) precursors are transported along the actin cables. The velocity of PSM precursors is diminished if Myo2p or Tpm1/2p function is impaired. Filamentous actin is not essential for meiotic progression, for shaping of the PSMs or for post-meiotic cytokinesis. However, actin is essential for spore wall formation. This requires the function of the Arp2/3p complex and involves large carbohydrate-rich compartments, which may be chitosome analogous structures. Mitotic cell division in baker's yeast Saccharomyces cerevisiae is accompanied by formation of a bud, which is connected to the mother cell by the bud neck. Subsequent division processes involve polarized growth of the plasma membrane in the bud and sequestration of cytoplasmic contents, including organelles and half of the nucleus into the daughter cell (1,2). A different morphogenetic programme, called sporulation, is performed during the meiotic cell divisions. The formation of the four meiotic progeny, the spores, occurs entirely in the cytoplasm of the mother cell (3-5).The new plasma membranes are formed from the socalled prospore membrane (PSM) precursors. The PSM precursors assemble at the spindle pole bodies (SPBs) in meiosis II to form the PSMs (6,7). Secretory vesicles and the exocyst are redirected towards the SPB and initiate PSM formation by homotypic vesicle fusion (8-10). Then the four PSMs grow out through the cytoplasm around the lobes of the nucleus. At the end of the meiotic divisions, after fission of the nuclear envelope, the PSMs close and each new compartment contains a haploid set of chromatids.Two structures specific for this process have been described: the meiotic plaque (MP) and the leading edge protein (LEP) coat [for a review, see (3)]. The MP of the SPBs has been demonstrated to be required for the initiation of membrane formation. It consists of at least three essential proteins (Mpc54p, Mpc70p, Spo74p), and one protein (Ady4p) with auxiliary function (7,11,12). In the absence of MPs, few PSM precursors bind to the SPBs, the others remain in the cytoplasm. These precursor structures are characterized by their content of the proteins Ssp1p, Ady3p and Don1p. Many precursor structures contain the t-SNAREs Sso1p and Sso2p as well (7,13). The proteins Ssp1, Ady3p and Don1p relocalize to the leading edge of the growing PSM after fusion of the precursor structures at the SPB. These proteins form the so-called LEP coat (13,14).The faithful and simultaneous completion ...

Precise control over organelle shapes is essential for cellular organization and morphogenesis. During yeast meiosis, prospore membranes (PSMs) constitute bell-shaped organelles that enwrap the postmeiotic nuclei leading to the cellularization of the mother cell's cytoplasm and to spore formation. Here, we analysed how the PSMs acquire their curved bell-shaped structure. We discovered that two antagonizing forces ensure PSM shaping and proper closure during cytokinesis. The Ssp1p-containing coat at the leading edge of the PSM generates a pushing force, which is counteracted by a novel pathway, the spore membranebending pathway (SpoMBe). Using genetics, we found that Sma2p and Spo1p, a phospholipase, as well as several GPIanchored proteins belong to the SpoMBe pathway. They exert a force all along the membrane, responsible for membrane bending during PSM biogenesis and for PSM closure during cytokinesis. We showed that the SpoMBe pathway involves asymmetric distribution of Sma2p and does not involve a GPI-protein-containing matrix. Rather, repulsive forces generated by asymmetrically distributed and dynamically moving GPI-proteins are suggested as the membrane-bending principle.