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The regulation of cellular membrane dynamics is crucial for maintaining proper cell growth and division. The Cdc48-Np14-Ufd1 complex is required for several regulated membrane-associated processes as part of the ubiquitin--proteasome system, including ER-associated degradation and the control of lipid composition in yeast. In this study we report the results of a genetic screen in Saccharomyces cerevisiae for extragenic suppressors of a temperature-sensitive np14 allele and the subsequent analysis of one suppressor, GET3/ARR4. The GET3 gene encodes an ATPase with homology to the regulatory component of the bacterial arsenic pump. Mutants of GET3 rescue several phenotypes of the np14 mutant and transcription of GET3 is coregulated with the proteasome, illustrating a functional relationship between GET3 and np14 in the ubiquitin--proteasome system. We have further found that Get3 biochemically interacts with the trans-membrane domain proteins Get1/Mdm39 and Get2/Rmd7 and that [DELTA]get3 is able to suppress phenotypes of get1 and get2 mutants, including sporulation defects. In combination, our characterization of GET3 genetic and biochemical interactions with np14, GET1, and GET2 implicates Get3 in multiple membrane-dependent pathways.

Spore formation in Saccharomyces cerevisiae involves the sequential deposition of multiple spore wall layers between the prospore membranes that surround each meiotic product. The Smk1p mitogen-activated protein (MAP) kinase plays a critical role in spore formation, but the proteins that interact with Smk1p to regulate spore morphogenesis have not been described. Using mass spectrometry, we identify Gsc2p as a Smk1p-associated protein. Gsc2p is a 1,3-[beta]-glucan synthase subunit involved in synthesizing an inner spore wall layer. We find that 1,3-[beta]-glucan synthase activity is elevated in sink1 mutants, suggesting that SMK1 negatively regulates GSC2. Although deposition of the two inner spore wall layers is normal in smk1 mutants, deposition of the outer layers is aberrant. However, eliminating GSC2 activity restores normal deposition of the third spore wall layer in smk1 mutants, indicating that negative regulation of GSC2 by SMK1 is important for spore wall deposition. Our findings suggest a model for the coordination of spore wall layer deposition in which Smk1p facilitates the transition between early and late phases of spore wall deposition by inhibiting a spore wall-synthesizing enzyme important for early phases of spore wall deposition. cell wall | sporulation | yeast | chitosan

Nuclear export of Far1p, a bifunctional protein required to stop the cell cycle and establish cell polarity in yeast mating, in response to pheromones is discussed. The process requires the export receptor Msn5p/Ste21p. It has been shown that Far1p acts in both subcellular compartments with nuclear Far1p necessary to the arrest of the cell cycle. Study results suggest nuclear export of Far1p by Msn5p/Ste21p coordinates the two separable functions of Far1p during mating.

Transcription factor, kinase and replication factor movements between nucleus and cytoplasm are important in activity control. The way in which phosphorylation promotes protein export from the nucleus is not well understood. The export of the yeast transcription factor Pho4 was investigated to determine how it is regulated in response to alterations in phosphate availability. The importin-beta-family member MSn5 is the shuttling receptor used by Pho4 for nuclear export.

The budding yeast Saccharomyces cerevisiae is a powerful model organism, partly because of the ease of genome alterations due to the combination of a fast generation time and many molecular genetic tools. Recent advances in CRISPR-based systems allow for the easier creation of alleles with internally inserted sequences within the coding regions of genes, such as the internal insertion of sequences that code for epitopes or fluorescent proteins. Here we briefly summarize some exisiting nomenclature standards and suggest nomenclature guidelines for internal insertion alleles which are informative, consistent, and computable., Competing Interests: The authors declare that there are no conflicts of interest present., (Copyright: © 2024 by the authors.)

Green fluorescent protein (GFP) has become an invaluable tool in biological research. Many GFP variants have been created that differ in brightness, photostability, and folding robustness. We have created two hybrid GFP variants, Envy and Ivy, which we placed in a vector for the C-terminal tagging of yeast proteins by PCR-mediated recombination. The Envy GFP variant combines mutations found in the robustly folding SuperfolderGFP and GFPγ, while the Ivy GFP variant is a hybrid of GFPγ and the yellow-green GFP variant, Clover. We compared Envy and Ivy to EGFP, SuperfolderGFP and GFPγ and found that Envy is brighter than the other GFP variants at both 30°C and 37°C, while Ivy is the most photostable. Envy and Ivy are recognized by a commonly used anti-GFP antibody, and both variants can be immunoprecipitated using the GFP TRAP Camelidae antibody nanotrap technology. Because Envy is brighter than the other GFP variants and is as photostable as GFPγ, we suggest that Envy should be the preferred GFP variant, while Ivy may be used in cases where photostability is of the utmost importance., (Copyright © 2015 John Wiley & Sons, Ltd.)