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

Abstract

In the filamentous fungus Sordaria macrospora (Sm), the STRIPAK complex is required for vegetative growth, fruiting-body development and hyphal fusion. 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 ∆pom33 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.

The vacuolar morphology protein VAC14 plays an important role in sexual development in the filamentous ascomycete Sordaria macrospora

“…To investigate vegetative hyphae and sexual structures, S. macrospora strains were grown on SWG-covered glass slides for 5 days or on solid SWG medium for 9 days under continuous light at 27 °C. The slides were prepared as described previously (Groth et al 2021 ), whereas SWG was used as solid medium and instead of liquid BMM water was poured into the petri dish to prevent desiccation of the growth medium. The documentation was performed with an AxioImage M1 microscope (Zeiss, Jena, Germany) using differential interference contrast (DIC) or a VHX-500F Digital Microscope (Keyence, Neu Isenburg, Germany).…”

“…For fluorescence microscopic analyses, S. macrospora strains were grown for 24 h on BMM-agar slides, as described in Groth et al ( 2021 ), for 72 h on solid SWG medium supplemented with 1.5% agarose (Biozym Scientific GmbH, Hessisch Oldendorf, Germany), or for 24–72 h on SWG + 1.5% agarose media supplemented with 0.1 M NaCl, 0.4 M sorbitol, 2.5 mM 3-AT, 0.003% SDS, 0.01% H 2 O 2 or without KNO 3 at 27 °C under continuous light conditions. To detect EGFP signals, Chroma filter set 49,002 (exciter ET470/40x, ET525/50 m, beamsplitter T495lpxr), for TagRFP-T/tdTomato/FM4-64-signals, Chroma filter set 49,005 (exciter ET545/30x, emitter ET620/60 m and beamsplitter T570LP) and for CMAC, Chroma filter set 49,000 (ET350/50x, emitter ET460/50 m and beamsplitter T400LP) was used.…”

“…Three biological replicates of each strain were analyzed in three independent experiments (n = 9). Asterisks (*) indicate a significant difference to the wt strain, according to Student's t-test (p < 0.05) ◂ (Groth et al 2021). For this, S. macrospora wt strains expressing either VAC14-TagRFP-T or POM33-EGFP were crossed.…”

Spatiotemporal Dynamic Regulation of Organelles During Meiotic Development, Insights From Fungi

“…In keeping with this hypothesis, a fraction of Rtn1 localizes to discrete puncta in the nuclear periphery during meiosis ( López-Fuentes et al, 2021 ). Interestingly, the putative S. macrospora nucleoporin Pom33 interacts with ER-shaping proteins, including a reticulon protein ( Groth et al, 2021 ). Although the location of this interaction is unknown, this could reflect a link between ER remodeling and the signaling pathways governing sexual development ( Groth et al, 2021 ; Kuck and Stein, 2021 ).…”

Special Issue “Signal Transductions in Fungi”

“…A well-characterized multi-subunit complex is the striatin-interacting phosphatases and kinases (STRIPAK) complex [ 22 ], for which a recently identified coiled-coil protein SCI1 was demonstrated to co-localize around the nucleus. Groth, Schmitt and co-workers [ 23 ] performed pulldown experiments with SCI, identifying a transmembrane nucleoporin as a potential nuclear anchor of STRIPAK. The authors speculate that this nucleoporin may temporarily anchor STRIPAK to the nuclear envelope.…”