A Large Nonconserved Region of the Tethering Protein Leashin Is Involved in Regulating the Position, Movement, and Function of Woronin Bodies in Aspergillus oryzae

Abstract

The Woronin body is a Pezizomycotina-specific organelle that is typically tethered to the septum, but upon hyphal wounding, it plugs the septal pore to prevent excessive cytoplasmic loss. Leashin (LAH) is a large Woronin body tethering protein that contains highly conserved N-and C-terminal regions and a long (ϳ2,500-amino-acid) nonconserved middle region. As the involvement of the nonconserved region in Woronin body function has not been investigated, here, we functionally characterized individual regions of the LAH protein of Aspergillus oryzae (AoLAH). In an Aolah disruptant, no Woronin bodies were tethered to the septum, and hyphae had a reduced ability to prevent excessive cytoplasmic loss upon hyphal wounding. Localization analysis revealed that the N-terminal region of AoLAH associated with Woronin bodies dependently on AoWSC, which is homologous to Neurospora crassa WSC (Woronin body sorting complex), and that the C-terminal region was localized to the septum. Elastic movement of Woronin bodies was observed when visualized with an AoLAH N-terminal-region-enhanced green fluorescent protein (EGFP) fusion protein. An N-and C-terminal fusion construct lacking the nonconserved middle region of AoLAH was sufficient for the tethering of Woronin bodies to the septum. However, Woronin bodies were located closer to the septum and exhibited impaired elastic movement. Moreover, expression of middle-region-deleted AoLAH in the Aolah disruptant did not restore the ability to prevent excessive cytoplasmic loss. These findings indicate that the nonconserved middle region of AoLAH has functional importance for regulating the position, movement, and function of Woronin bodies.

A novel Pezizomycotina‐specific protein with gelsolin domains regulates contractile actin ring assembly and constriction in perforated septum formation

“…Woronin bodies were visualized by expressing the fusion protein AoHex1‐EGFP (Figure c). In accordance with the previous studies (Han et al, ; Maruyama et al, ), Woronin bodies were tethered to septa at both sides near the pore in wild type and in normal septa in Δ glpA (Figure c). On the other hand, morphologically abnormal septa in Δ glpA had aberrant tethering of Woronin bodies (Figure c).…”

“…Therefore, using actin‐related gene ontology and BLAST‐based selection, we identified a novel Pezizomycotina‐specific gelsolin‐like protein GlpA and characterized its function. Aspergillus oryzae was selected in this study as a representative of Pezizomycotina species, since septal pore regulation has been extensively characterized by an experimental technique of quantitatively analyzing the ability to protect the adjacent cells upon hyphal wounding and by microscopic observation of the septum‐localizing proteins (Bleichrodt et al, ; Escaño et al, ; Han, Jin, Maruyama, & Kitamoto, ; Huang, Maruyama, & Kitamoto, ; Maruyama, Escaño, & Kitamoto, ; Maruyama et al, ; Tsukasaki, Saeki, Katayama, Maruyama, & Kitamoto, ). We have shown that GlpA is required for proper septum formation frequency, normal septum morphology and septal pore function by participating in CAR assembly and constriction.…”

Recognition of the polycistronic nature of human genes is critical to understanding the genotype-phenotype relationship

“…Cold Spring Harbor Laboratory Press on June 7, 2019 -Published by genome.cshlp.org Downloaded from accordingly, the possibility to identify nonconserved yet functional proteins arouses strong opinions (The ENCODE Project Consortium 2012; Graur et al 2013;Han et al 2014). Available online tools for visualization of ribosome profiling data are listed in Table 2. Evidence at the protein level Mass spectrometry (MS)-based proteomics has emerged as the gold standard technique to assess the protein landscape of a cell or tissue and thus can offer additional evidence beyond ribosome profiling Mann 2003, 2016;Vogel and Marcotte 2012;Huttlin et al 2015).…”

Cell biology of the Koji mold Aspergillus oryzae

“…8). 11,15,31) We cloned the Aohex1 gene, which encodes a protein constituting the Woronin body, and determined that the AoHex1 multimer is formed in peroxisomes and plays an essential role in survival against catastrophic cell injury. 32) It is important to note that the septal pore is not constitutively open and that fluctuations between the open-closed state is dependent on the behavior of the AoSO protein in response to environmental stress.…”