Age-based inheritance of centrosomes in eukaryotic cells is associated with faithful chromosome distribution in asymmetric cell divisions. During ascospore formation, such an inheritance mechanism targets the yeast centrosome equivalents, the spindle pole bodies (SPBs) at meiosis II onset. Decreased nutrient availability causes initiation of spore formation at only the younger SPBs and their associated genomes. This mechanism ensures encapsulation of nonsister genomes, which preserves genetic diversity and provides a fitness advantage at the population level. Here, by usage of an enhanced system for sporulation-induced protein depletion, we demonstrate that the core mitotic exit network (MEN) is involved in age-based SPB selection. Moreover, efficient genome inheritance requires Dbf2/20-Mob1 during a late step in spore maturation. We provide evidence that the meiotic functions of the MEN are more complex than previously thought. In contrast to mitosis, completion of the meiotic divisions does not strictly rely on the MEN whereas its activity is required at different time points during spore development. This is reminiscent of vegetative MEN functions in spindle polarity establishment, mitotic exit, and cytokinesis. In summary, our investigation contributes to the understanding of age-based SPB inheritance during sporulation of and provides general insights on network plasticity in the context of a specialized developmental program. Moreover, the improved system for a developmental-specific tool to induce protein depletion will be useful in other biological contexts.

23The ubiquitin-proteasome system (UPS) controls cellular functions by maintenance of a 24 functional proteome and degradation of key regulatory proteins. Central to the UPS is the 25 proteasome that adjusts the abundance of numerous proteins, thereby safeguarding their 26 activity or initiating regulatory events. Here, we demonstrate that the essential 27Saccharomyces cerevisiae protein Yjr141w/Ipa1 (Important for cleavage and 28 PolyAdenylation) belongs to the HECT_2 (homologous to E6-AP carboxyl terminus_2) 29 family. We found that five cysteine residues within the HECT_2 family signature and the C-30 terminus are essential for Ipa1 activity. Furthermore, Ipa1 interacts with several ubiquitin-31 conjugating enzymes in vivo and localizes to the cytosol and nucleus. Importantly, Ipa1 32 has impact on proteasome activity, which is indicated by the activation of the Rpn4 regulon 33 as well as by decreased turnover of destabilized proteasome substrates in an IPA1 mutant. 34These changes in proteasome activity might be connected to reduced maturation or 35 modification of proteasomal core particle proteins. Our results highlight the influence of 36Ipa1 on the UPS. The conservation within the HECT_2 family and the connection of the 37 human HECT_2 family member to an age-related degeneration disease might suggest that 38

Regulation of protein stability is a fundamental process in eukaryotic cells and pivotal to, e.g., cell cycle progression, faithful chromosome segregation, or protein quality control. Synthetic regulation of protein stability requires conditional degradation sequences (degrons) that induce a stability switch upon a specific signal. Fusion to a selected target protein permits to influence virtually every process in a cell. Light as signal is advantageous due to its precise applicability in time, space, quality, and quantity. Light control of protein stability was achieved by fusing the LOV2 photoreceptor domain of Arabidopsis thaliana phototropin1 with a synthetic degron (cODC1) derived from the carboxy-terminal degron of ornithine decarboxylase to obtain the photosensitive degron (psd) module. The psd module can be attached to the carboxy terminus of target proteins that are localized to the cytosol or nucleus to obtain light control over their stability. Blue light induces structural changes in the LOV2 domain, which in turn lead to activation of the degron and thus proteasomal degradation of the whole fusion protein. Variants of the psd module with diverse characteristics are useful to fine-tune the stability of a selected target at permissive (darkness) and restrictive conditions (blue light).

In the yeast Saccharomyces cerevisiae and other ascomycetes, the maintenance of cell wall integrity is governed by a family of plasma-membrane spanning sensors that include the Wsc-type proteins. These cell wall proteins apparently sense stress-induced mechanical forces at the cell surface and target the cell wall integrity (CWI) signaling pathway, but the structural base for their sensor function is yet unknown. Here, we solved a high-resolution crystal structure of the extracellular cysteine-rich domain (CRD) of yeast Wsc1, which shows the characteristic PAN/Apple domain fold with two of the four Wsc1 disulfide bridges being conserved in other PAN domain cores. Given the general function of PAN domains in mediating protein–protein and protein–carbohydrate interactions, this finding underpins the importance of Wsc domains in conferring sensing and localization functions. Our Wsc1 CRD structure reveals an unusually high number of surface-exposed aromatic residues that are conserved in other fungal CRDs, and can be arranged into three solvent-exposed clusters. Mutational analysis demonstrates that two of the aromatic clusters are required for conferring S. cerevisiae Wsc1-dependent resistance to the glucan synthase inhibitor caspofungin, and the chitin-binding agents Congo red and Calcofluor white. These findings suggest an essential role of surface-exposed aromatic clusters in fungal Wsc-type sensors that might include an involvement in stress-induced sensor-clustering required to elicit appropriate cellular responses via the downstream CWI pathway.