Analysis of the Putative Nucleoporin POM33 in the Filamentous Fungus Sordaria macrospora.

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The SmSTRIPAK core consists of the striatin homolog PRO11, the scaffolding subunit of phosphatase PP2A, SmPP2AA, and its catalytic subunit SmPP2Ac1. Among other STRIPAK proteins, the recently identified coiled-coil protein SCI1 was demonstrated to co-localize around the nucleus. Pulldown experiments with SCI identified the transmembrane nucleoporin (TM Nup) SmPOM33 as a potential nuclear-anchor of SmSTRIPAK. Localization studies revealed that SmPOM33 partially localizes to the nuclear envelope (NE), but mainly to the endoplasmic reticulum (ER). We succeeded to generate a Δpom33 deletion mutant by homologous recombination in a new S. macrospora Δku80 recipient strain, which is defective in non-homologous end joining. Deletion of Smpom33 did neither impair vegetative growth nor sexual development. In pulldown experiments of SmPOM33 followed by LC/MS analysis, ER-membrane proteins involved in ER morphology, protein translocation, glycosylation, sterol biosynthesis and Ca2+-transport were significantly enriched. Data are available via ProteomeXchange with identifier PXD026253. Although no SmSTRIPAK components were identified as putative interaction partners, it cannot be excluded that SmPOM33 is involved in temporarily anchoring the SmSTRIPAK to the NE or other sites in the cell.", "claims": [{"id": "claim-method", "statement": "Data were evaluated in a semiquantitative method with label-free quantification (LFQ) based on signal intensities using the MaxQuant and Perseus programs [52,54]. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [100] partner repository with the dataset identifier PXD026253.", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:34575720", "locator": "sections[19] \"3.5. 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Strains, Media and Growth Conditions", "row_count": 28, "column_count": 3}, "table": {"label": "Table 1", "caption": "Strains used and generated in this study.", "rows": [["Strain", "Genotype", "References"], ["Escherichia coli"], ["MACH1", "ΔrecA1398, endA1, tonA, Φ80ΔlacM15, ΔlacX74, hsdR, (rK-mK+)", "Invitrogen"], ["Saccharomyces cerevisiae"], ["PJ69-4A", "MATa, trp1-901, leu2-3, 112, ura3-52, his3-200, gal4Δ, gal80Δ, LYS2::GAL1-HIS3, GAL2-ADE2, met2::GAL7-lacZ", "[35]"], ["Sordaria macrospora"], ["DSM997", "wild type (wt)", "DSMZ"], ["S23442", "mutation in fus1-1 gene, brownish ascospores, fertile", "[41]"], ["Δku70", "Δku70::natR, fertile", "[42]"], ["Δsci1", "Δsci1::hygR, ssi, sterile", "[14]"], ["Δsci1::nca1-TagRFP-Tect", "ectopic integration of p5′nca1-TagRFP-T _nat into Δsci1; hygR, natR pt, sterile,Pnca1::nca1::TagRFP-T::TtrpC", "Reschka and Pöggeler (unpublished)"], ["wt::egfpect", "ectopic integration of p1783-1 into DSM997; hygR, ssi, fertile, Pgpd::egfp::TtrpC", "[43]"], ["wt::TagRFP-Tect", "ectopic integration of pTagRFP-T_nat into DSM997; natR, ssi, fertile, Pccg1::TagRFP-T::TtrpC", "[44]"], ["wt::GH2Aect", "ectopic integration of pGH2A into DSM997; hygR, pt, fertile, Pgpd::hh2a::egfp::TtrpC", "Reschka and Pöggeler (unpublished)"], ["fus::RH2Bect", "ectopic integration of pRH2B in S23442; hygR, pt, fertile, Pgpd::hh2b::tdTomato::TtrpC", "Reschka and Pöggeler (unpublished)"], ["wt::nca1-TagRFP-Tect", "ectopic integration of p5′nca1-TagRFP-T_hyg into DSM997; hygR, pt, fertile,Pnca1::nca1::TagRFP-T::TtrpC", "Werner and Pöggeler (unpublished)"], ["Δku80", "Δku80::hygR, ssi, fertile", "This study"], ["Δpom33", "Δpom33::natR, ssi, fertile", "This study"], ["wt::pom33-TagRFP-Tect", "ectopic integration of p5′pom33-TagRFP-T_nat into DSM997; natR, ssi, fertile,Ppom33::pom33:: TagRFP-T::TtrpC", "This study"], ["wt::pom33-TagRFP-Tect", "ectopic integration of p5′pom33-TagRFP-T_hyg into DSM997; hygR, pt, fertile,Ppom33::pom33:: TagRFP-T::TtrpC", "This study"], ["wt::pom33-egfpect", "ectopic integration of p5′pom33-egfp into DSM997; natR, ssi, fertile,Ppom33::pom33::egfpt::TtrpC", "This study"], ["wt::pom33-TagRFP-Tect + pom152-egfpect", "ectopic integration of p5′pom33-TagRFP-T_hyg and pSmPOM152GFP into DSM997; hygR, natR, pt, fertile,Ppom33::pom33:: TagRFP-T::TtrpC; Pccg1::pom152::egfp::TtrpC", "This study"], ["wt::pom33-TagRFP-Tect + sci1-egfpect", "ectopic integration of p5′pom33-TagRFP-T_hyg and p5′sci1gfp_nat into DSM997; hygR, natR, pt, fertile,Ppom33::pom33:: TagRFP-T::TtrpC; Psci1::sci1::egfp::TtrpC", "This study"], ["Δpom33::pom33-TagRFP-Tect", "ectopic integration of p5′pom33-TagRFP-T_hyg into Δpom33; hygR, natR ssi, fertile,Ppom33::pom33:: TagRFP-T::TtrpC", "This study"], ["Δpom33::RH2Bect", "ectopic integration of pRH2B into Δpom33; hygR, natR pt, fertile,Pgpd::hh2b::tdTomato::TtrpC", "This study"], ["Δpom33:: nca1-TagRFP-Tect", "ectopic integration of p5′nca1-TagRFP-T_hyg into Δpom33; hygR, natR pt, fertile,Pnca1::nca1::TagRFP-T::TtrpC", "This study"], ["Δpom33::sci1-egfpect", "ectopic integration of p5′sci1-egfp_hyg into Δpom33; hygR, natR pt, fertile,Psci1::sci1::egfp::TtrpC", "This study"], ["Δpom33::pro11-egfpect", "ectopic integration of pPRO11-GFP_hyg into Δpom33; hygR, natR pt, fertile,Pccg1::HA::pro11::egfp::TtrpC", "This study"]], "row_count": 28, "column_count": 3, "numeric_cells": 43, "header_rows": 1, "locator": "section[3]/table-wrap[1]#jof-07-00682-t001"}}], "gaps": ["asset:data:geo:PXD026253 not downloaded", "asset:data:geo:PXD026253 licence not read"]}, "analysis": {"state": "partial", "methods": [{"id": "method-1", "family": null, "specific": null, "label": "Label-free quantification (LFQ)", "dsl_operations": [], "missing_operations": [], "expressible": null, "parameters": {}, "data_shapes": [], "evidence": {"score": 1.0, "band": "reordered", "quote": "Data were evaluated in a semiquantitative method with label-free quantification (LFQ) based on signal intensities using the MaxQuant and Perseus programs [52,54]. 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