A reporter system to study unconventional secretion of proteins avoiding N-glycosylation in Ustilago maydis

Abstract

Unconventional secretion of proteins in eukaryotes is characterized by the circumvention of the Endoplasmic Reticulum (ER). As a consequence proteins exported by unconventional pathways lack N -glycosylation, a post-transcriptional modification that is initiated in the ER during classical secretion. We are exploiting the well-established enzyme β-glucuronidase (GUS) to assay unconventional protein secretion (UPS). This bacterial protein is perfectly suited for this purpose because it carries a eukaryotic N -glycosylation motif. Modification of this residue by attachment of sugar moieties during the passage of the ER apparently causes a very strong reduction in GUS activity. Hence, this enzyme can only be secreted in an active state, if the export mechanism does not involve ER passage. Here, we describe a reporter system applied in the corn smut fungus Ustilago maydis that is based on this observation and can be used to test if candidate proteins are secreted to the culture supernatant via alternative pathways avoiding N -glycosylation. Importantly, this system is the basis for the establishment of genetic screens providing mechanistic insights into unknown UPS pathways in the future.