A Reporter System to Study Unconventional Secretion of Proteins Avoiding N-Glycosylation in Ustilago maydis
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.
Key wordsβ-Glucuronidase (GUS) Unconventional protein secretion (UPS) 4-Methylumbelliferyl-β-D-glucuronide (MUG) N-Glycosylation Ustilago maydis
We are grateful to Drs. Janine Koepke, Saskia Kreibich, Thomas Brefort, and Regine Kahmann for their input and support, and to Dr. Michael Feldbrügge for critical discussions, comments on the manuscript, and general support. J.S. and M.T. received a fellowship of the Graduate School of the Cluster Industrial Biotechnology 2021 (CLIB-GC) and K.S. was supported by the Strategic Research Fund of the Heinrich-Heine University Düsseldorf. The scientific activities of the Bioeconomy Science Center were supported financially by the Ministry of Innovation, Science and Research within the framework of the NRW Strategieprojekt BioSC (No. 313/323‐400‐002 13).
- 14.Kämper J, Kahmann R, Bölker M, Ma LJ, Brefort T, Saville BJ, Banuett F, Kronstad JW, Gold SE, Müller O, Perlin MH, Wösten HA, de Vries R, Ruiz-Herrera J, Reynaga-Pena CG, Snetselaar K, McCann M, Pérez-Martin J, Feldbrügge M, Basse CW, Steinberg G, Ibeas JI, Holloman W, Guzman P, Farman M, Stajich JE, Sentandreu R, Gonzalez-Prieto JM, Kennell JC, Molina L, Schirawski J, Mendoza-Mendoza A, Greilinger D, Münch K, Rössel N, Scherer M, Vranes M, Ladendorf O, Vincon V, Fuchs U, Sandrock B, Meng S, Ho EC, Cahill MJ, Boyce KJ, Klose J, Klosterman SJ, Deelstra HJ, Ortiz-Castellanos L, Li W, Sanchez-Alonso P, Schreier PH, Hauser-Hahn I, Vaupel M, Koopmann E, Friedrich G, Voss H, Schlüter T, Margolis J, Platt D, Swimmer C, Gnirke A, Chen F, Vysotskaia V, Mannhaupt G, Güldener U, Münsterkotter M, Haase D, Oesterheld M, Mewes HW, Mauceli EW, DeCaprio D, Wade CM, Butler J, Young S, Jaffe DB, Calvo S, Nusbaum C, Galagan J, Birren BW (2006) Insights from the genome of the biotrophic fungal plant pathogen Ustilago maydis. Nature 444(7115):97–101. doi: 10.1038/nature05248 CrossRefPubMedGoogle Scholar