Specific phosphoinositide interaction of Jps1 is a key feature during unconventional secretion in Ustilago maydis.

Protein secretion is indispensable for essential cellular processes in eukaryotic cells, contributing significantly to nutrient acquisition, defense, or communication. Alternative pathways bypassing the endomembrane system collectively referred to as unconventional secretion are gaining increasing attention. A number of important molecules such as cytokines, fibroblast growth factor, or viral proteins are being exported through these mechanistically diverse pathways. In the fungal model Ustilago maydis, cytokinesis-dependent unconventional secretion mediates export of the chitinase Cts1 via the fragmentation zone. This membrane-rich compartment is formed between mother and daughter cells during cytokinesis. Recently, we identified Jps1, a previously uncharacterized protein, as a crucial factor for Cts1 localization and export. Combining biochemical experiments and in vivo studies, we here uncover two pivotal features of Jps1: dimerization and phosphatidylinositol phosphate (PIP) binding. Our findings reveal that a conserved structural core domain mediates homodimerization, while surrounding flexible variable regions suggest potential diversification in different basidiomycete species. Jps1 does not harbor a canonical PIP-binding domain, but instead specificity of the interaction with the preferred PIP PI(4,5)P is determined by basic residues. Importantly, loss of PI(4,5)P-binding specificity results in mislocalization, morphological defects, and reduced extracellular Cts1 activity, particularly at low cell densities. Our discoveries shed light on previously unknown key features of Jps1 and represent a crucial step towards understanding the broader implications of unconventional secretion in eukaryotic cells.