Filamin FLN-2 promotes MVB biogenesis by mediating vesicle docking on the actin cytoskeleton.

Multivesicular bodies (MVBs) contain intralumenal vesicles that are delivered to lysosomes for degradation or released extracellularly for intercellular signaling. Here, we identified Caenorhabditis elegans filamin FLN-2 as a novel regulator of MVB biogenesis. FLN-2 co-localizes with V-ATPase subunits on MVBs, and the loss of FLN-2 affects MVB biogenesis, reducing the number of MVBs in C. elegans hypodermis. FLN-2 associates with actin filaments and is required for F-actin organization. Like fln-2(lf) mutation, inactivation of the V0 or V1 sector of V-ATPase or inhibition of actin polymerization impairs MVB biogenesis. Super-resolution imaging shows that FLN-2 docks V-ATPase-decorated MVBs onto actin filaments. FLN-2 interacts via its calponin-homology domains with F-actin and the V1-E subunit, VHA-8. Our data suggest that FLN-2 mediates the docking of MVBs on the actin cytoskeleton, which is required for MVB biogenesis.

VerZ, a Zn(II)2Cys6 DNA-binding protein, regulates the biosynthesis of verticillin in Clonostachys rogersoniana.

Verticillins are the dimeric epipolythiodioxopiperazines (ETPs) produced by the fungus Clonostachys rogersoniana. Despite their profound biological effects, they are commonly produced in rice medium as complex mixtures that are difficult to separate, limiting further study and evaluation for this class of metabolites. Therefore, there is an urgent need to understand the regulation of verticillin biosynthesis. Recently, we cloned the biosynthetic gene cluster of verticillin (ver), and identified the only regulatory gene verZ in this cluster. The deduced product of verZ contains a basic Zn(II)2Cys6 DNA-binding domain. Disruption of verZ significantly reduced the production of 11'-deoxyverticillin A (C42) and decreased the transcriptional level of the verticillin biosynthetic genes. To further reveal its function, a recombinant gene encoding the DNA-binding domain of VerZ was expressed in E. coli and the His6-tagged VerZbd was purified to homogeneity by Ni-NTA chromatography. Electrophoretic mobility shift assays (EMSAs) showed that VerZbd bound specifically to the promoter regions of the verticillin biosynthetic genes. Bioinformatic analysis of the VerZbd-binding regions revealed a conserved palindromic sequence of (T/C)(C/A)(G/T)GN3CC(G/T)(A/G)(G/C). Base substitution of the conserved sequence completely abolished the binding activity of VerZbd to its targets. These results suggested that VerZ controls verticillin production through directly activating transcription of the biosynthetic genes in C. rogersoniana.

Functional analysis of the selective autophagy related gene Acatg11 in Acremonium chrysogenum.

Autophagy is a highly conserved degradation system in eukaryotes. Selective autophagy is used for the degradation of selective cargoes. Selective autophagic processes of yeast include pexophagy, mitophagy, and cytoplasm-to-vacuole targeting (Cvt) pathway in which particular vacuolar proteins, such asaminopeptidase I (Ape1), are selectively transported to vacuoles. However, the physiological role of selective autophagy remains elusive in filamentous fungi. ATG11 family proteins asa basic scaffold are essential for most selective autophagy pathways in yeast. Here, Acatg11, encoding a putative ATG11 family protein, was identified and cloned from the cephalosporin producing strain Acremonium chrysogenum based on the sequence similarity of ATG11 superfamily proteins. Disruption of Acatg11 inhibited the maturation of preApe1 during fermentation indicating that Acatg11 is involved in Cvt pathway. In addition, pexophagy and mitophagy were blocked in the Acatg11 disruption mutant (ΔAcatg11). Intriguingly, the nonselective autophagy was deficient in ΔAcatg11 under starvation induction or during fermentation. Disruption of Acatg11 significantly enhanced fungal conidiation, but reduced cephalosporin production. These results indicated that Acatg11 is required for both selective and nonselective autophagy during fermentation and has a strong impact on morphological differentiation and cephalosporin production of A. chrysogenum.