The unconventional secretion of PepN is independent of a functional autophagy machinery in the filamentous fungus Aspergillus niger.
During unconventional protein secretion (UPS), proteins do not pass through the classical endoplasmic reticulum (ER)-Golgi-dependent pathway, but are transported to the cell membrane via alternative routes. One type of UPS is dependent on several autophagy-related (Atg) proteins in yeast and mammalian cells, but mechanisms for unconventional secretion are largely unknown for filamentous fungi. In this study, we investigated whether the autophagy machinery is used for UPS in the filamentous fungus Aspergillus niger An aspartic protease, which we called PepN, was identified as being likely to be secreted unconventionally, as this protein is highly abundant in culture filtrates during carbon starvation while it lacks a conventional N-terminal secretion sequence. We analysed the presence of PepN in the culture filtrates of carbon starved wild-type, atg1 and atg8 deletion mutant strains by Western blot analysis and by secretome analysis using nanoLC-ESI-MS/MS (wild-type and atg8 deletion mutant). Besides the presence of carbohydrate-active enzymes and other types of proteases, PepN was abundantly found in culture filtrates of both wild-type and atg deletion strains, indicating that the secretion of PepN is independent of the autophagy machinery in A. niger and hence most likely occurs via a different mechanism.
Leiden University, Institute of Biology Leiden, Molecular Microbiology and Biotechnology, Sylviusweg 72, 2333 BE Leiden, The Netherlands.
Anne-Marie
AM
Burggraaf
Leiden University, Institute of Biology Leiden, Molecular Microbiology and Biotechnology, Sylviusweg 72, 2333 BE Leiden, The Netherlands Dutch DNA Biotech, Utrechtseweg 48, 3704 HE, Zeist, the Netherlands.
Peter J
PJ
Punt
Leiden University, Institute of Biology Leiden, Molecular Microbiology and Biotechnology, Sylviusweg 72, 2333 BE Leiden, The Netherlands a.f.j.ram@biology.leidenuniv.nl.
Arthur F J
AF
Ram
D001234:Aspergillus niger / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D001343:Autophagy; D000071190:Autophagy-Related Protein 8 Family / Q000235:genetics / Q000378:metabolism; D002244:Carbon / Q000378:metabolism; D003470:Culture Media / Q000737:chemistry; D005656:Fungal Proteins / Q000378:metabolism*; D021381:Protein Transport; D020543:Proteome; D017384:Sequence Deletion; D053719:Tandem Mass Spectrometry
D000071190:Autophagy-Related Protein 8 Family; D003470:Culture Media; D005656:Fungal Proteins; D020543:Proteome; D002244:Carbon
Atg1; Atg8; protease activity; proteome analysis; secretome