Intrinsically disordered proteins aggregate at fungal cell-to-cell channels and regulate intercellular connectivity.

Like animals and plants, multicellular fungi possess cell-to-cell channels (septal pores) that allow intercellular communication and transport. Here, using a combination of MS of Woronin body-associated proteins and a bioinformatics approach that identifies related proteins based on composition and character, we identify 17 septal pore-associated (SPA) proteins that localize to the septal pore in rings and pore-centered foci. SPA proteins are not homologous at the primary sequence level but share overall physical properties with intrinsically disordered proteins. Some SPA proteins form aggregates at the septal pore, and in vitro assembly assays suggest aggregation through a nonamyloidal mechanism involving mainly α-helical and disordered structures. SPA loss-of-function phenotypes include excessive septation, septal pore degeneration, and uncontrolled Woronin body activation. Together, our data identify the septal pore as a complex subcellular compartment and focal point for the assembly of unstructured proteins controlling diverse aspects of intercellular connectivity.

Annexin B9 binds to ß(H)-spectrin and is required for multivesicular body function in Drosophila.

The role of the cytoskeleton in protein trafficking is still being defined. Here, we describe a relationship between the small Ca(2+)-dependent membrane-binding protein Annexin B9 (AnxB9), apical ß(Heavy)-spectrin (ß(H)) and the multivesicular body (MVB) in Drosophila. AnxB9 binds to a subset of ß(H) spliceoforms, and loss of AnxB9 results in an increase in basolateral ß(H) and its appearance on cytoplasmic vesicles that overlap with the MVB markers Hrs, Vps16 and EPS15. Similar colocalizations are seen when ß(H)-positive endosomes are generated either by upregulation of ß(H) in pak mutants or through the expression of the dominant-negative version of ß(H). In common with other mutations disrupting the MVB, we also show that there is an accumulation of ubiquitylated proteins and elevated EGFR signaling in the absence of AnxB9 or ß(H). Loss of AnxB9 or ß(H) function also causes the redistribution of the DE-Cadherin (encoded by shotgun) to endosomal vesicles, suggesting a rationale for the previously documented destabilization of the zonula adherens in karst (which encodes ß(H)) mutants. Reduction of AnxB9 results in degradation of the apical-lateral boundary and the appearance of the basolateral proteins Coracle and Dlg on internal vesicles adjacent to ß(H). These results indicate that AnxB9 and ß(H) are intimately involved in endosomal trafficking to the MVB and play a role in maintaining high-fidelity segregation of the apical and lateral domains.