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      The Smk1 MAPK and Its Activator, Ssp2, Are Required for Late Prospore Membrane Development in Sporulating Saccharomyces cerevisiae

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          Abstract

          During sporulation in the budding yeast Saccharomyces cerevisiae, proper development of the prospore membrane is necessary for the formation of viable spores. The prospore membrane will eventually become the plasma membrane of the newly formed haploid spore and also serves as the template for the deposition of the spore wall. The prospore membrane is generated de novo during meiosis II and the growing edge of the prospore membrane is associated with the Leading Edge Protein (LEP) complex. We find that the Smk1 MAP kinase, along with its activator Ssp2, transiently localizes with the LEP during late meiosis II. SSP2 is required for the leading edge localization of Smk1; this localization is independent of the activation state of Smk1. Like other LEP components, the localization of Smk1 at the leading edge also depends on Ady3. Although prospore membrane development begins normally in smk1 and ssp2 mutants, late prospore membrane formation is disrupted, with the formation of ectopic membrane compartments. Thus, MAP kinase signaling plays an important role in the formation of the prospore membrane.

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          Most cited references33

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          Fiji: an open-source platform for biological-image analysis.

          Fiji is a distribution of the popular open-source software ImageJ focused on biological-image analysis. Fiji uses modern software engineering practices to combine powerful software libraries with a broad range of scripting languages to enable rapid prototyping of image-processing algorithms. Fiji facilitates the transformation of new algorithms into ImageJ plugins that can be shared with end users through an integrated update system. We propose Fiji as a platform for productive collaboration between computer science and biology research communities.
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            Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae.

            An important recent advance in the functional analysis of Saccharomyces cerevisiae genes is the development of the one-step PCR-mediated technique for deletion and modification of chromosomal genes. This method allows very rapid gene manipulations without requiring plasmid clones of the gene of interest. We describe here a new set of plasmids that serve as templates for the PCR synthesis of fragments that allow a variety of gene modifications. Using as selectable marker the S. cerevisiae TRP1 gene or modules containing the heterologous Schizosaccharomyces pombe his5+ or Escherichia coli kan(r) gene, these plasmids allow gene deletion, gene overexpression (using the regulatable GAL1 promoter), C- or N-terminal protein tagging [with GFP(S65T), GST, or the 3HA or 13Myc epitope], and partial N- or C-terminal deletions (with or without concomitant protein tagging). Because of the modular nature of the plasmids, they allow efficient and economical use of a small number of PCR primers for a wide variety of gene manipulations. Thus, these plasmids should further facilitate the rapid analysis of gene function in S. cerevisiae.
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              Improved Blue, Green, and Red Fluorescent Protein Tagging Vectors for S. cerevisiae

              Fluorescent protein fusions are a powerful tool to monitor the localization and trafficking of proteins. Such studies are particularly easy to carry out in the budding yeast Saccharomyces cerevisiae due to the ease with which tags can be introduced into the genome by homologous recombination. However, the available yeast tagging plasmids have not kept pace with the development of new and improved fluorescent proteins. Here, we have constructed yeast optimized versions of 19 different fluorescent proteins and tested them for use as fusion tags in yeast. These include two blue, seven green, and seven red fluorescent proteins, which we have assessed for brightness, photostability and perturbation of tagged proteins. We find that EGFP remains the best performing green fluorescent protein, that TagRFP-T and mRuby2 outperform mCherry as red fluorescent proteins, and that mTagBFP2 can be used as a blue fluorescent protein tag. Together, the new tagging vectors we have constructed provide improved blue and red fluorescent proteins for yeast tagging and three color imaging.
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                Author and article information

                Journal
                J Fungi (Basel)
                J Fungi (Basel)
                jof
                Journal of Fungi
                MDPI
                2309-608X
                14 January 2021
                January 2021
                : 7
                : 1
                : 53
                Affiliations
                Department of Biology, University of Massachusetts Boston, 100 Morrisey Boulevard, Boston, MA 02125, USA; Matthew.Durant001@ 123456umb.edu (M.D.); joseph.roesner@ 123456merck.com (J.M.R.); xheni.mucelli001@ 123456umb.edu (X.M.); cslubowski@ 123456generationbio.com (C.J.S.); Erin.Klee001@ 123456umb.edu (E.K.); Brian.Seitz001@ 123456umb.edu (B.C.S.); wallisz@ 123456bc.edu (Z.W.)
                Author notes
                [* ]Correspondence: linda.huang@ 123456umb.edu
                [†]

                These authors contributed equally to this work.

                [‡]

                Present Affiliation: Merck, 33 Avenue Louis Pasteur, Boston, MA 02115, USA.

                [§]

                Present Affiliation: Generation Bio, 301 Binney Street, Cambridge, MA 02124, USA.

                [‖]

                Present Affiliation: Department of Biology, Boston College, 140 Commonwealth Avenue, Chestnut Hill, MA 02467, USA.

                Author information
                https://orcid.org/0000-0003-1901-1052
                https://orcid.org/0000-0003-0257-3198
                https://orcid.org/0000-0002-9033-4391
                Article
                jof-07-00053
                10.3390/jof7010053
                7828665
                33466572
                68357b75-412e-4026-b450-dd74f5abc0c2
                © 2021 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 13 November 2020
                : 09 January 2021
                Categories
                Article

                saccharomyces cerevisiae,sporulation,prospore membrane,meiosis ii,map kinase signaling,leading edge protein complex

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