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The FlbA-regulated predicted transcription factor rpnR of Aspergillus niger is involved in protein secretion and stress resistance

ABSTRACT: Proteins are secreted throughout the mycelium of Aspergillus niger except for the sporulating zone. Inactivation of flbA results in colonies that do not reproduce asexually and secrete proteins throughout the colony. Moreover, ΔflbA hyphae lyse and have thinner cell walls. This pleiotropic phenotype is associated with differential expression of 38 transcription factor genes, of which rpnR was inactivated in this study. Biomass formation and sporulation was not affected in strain ΔrpnR but protein secretion was severely reduced. Moreover, ΔrpnR showed decreased resistance to H2O2 and the unfolded protein response inducing agent dithiothreitol. The secretomes of ΔrpnR and wild type consisted of 342 and 367 proteins, respectively. Secretion of several CAZymes and proteins involved in cell wall remodeling was more strongly reduced when compared to other proteins within the secretome of ΔrpnR. Whole genome expression analysis of xylose-grown colonies showed that RpnR activates expression of ribosomal subunits as well as small secreted proteins. On the other hand, it inhibited expression of the genes encoding the arabinofuranosidases AbfA, AbfB and AbfE, as well as genes related to amino acid metabolism, xenobiotics biodegradation and metabolism, lipid metabolism, and metabolism of cofactors and vitamins. Taken together, RpnR is involved in protein synthesis and may also be involved in cell wall architecture. Both functions could explain the reduced protein secretion and proteotoxic stress resistance in the deletion strain.

ORGANISM(S): Aspergillus niger

PROVIDER: GSE102899 | GEO | 2018/06/01

REPOSITORIES: GEO

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Project description:Protein secretion is an essential process in cell biology. The conventional secretion pathway is well known, and involves proteins with a signal sequence being directed into the endoplasmic reticulum. Once in the lumen of the endoplasmic reticulum, proteins are trafficked through the Golgi and remain separate from the cytosol throughout their journey to the outside of the cell. More recently, proteins without a signal sequence have been found outside the cell. These proteins are secreted unconventionally, and avoid the ER-Golgi route. For both pathways, some aspects remain unclear, particularly surrounding the regulation of protein secretion. Galectin-3, an unconventionally secreted protein, was used here to investigate both conventional and unconventional protein secretion. The unconventional protein secretion of galectin-3 is poorly understood. To address this, genetic manipulation of galectin-3 was used to gain insights into the mechanism of galectin-3 secretion. It was found that galectin-3 does not require unfolding or binding to cell surface glycoproteins to be secreted. Galectin-3 was also used to assess protein secretion using a genome-wide CRISPR screen. As galectin‑3 binds to cell surface glycoproteins, galectin-3 on the cell surface was used to assess secretion of these glycoproteins. Hits were validated by a glycoprotein secretion assay. Using this pipeline, 93 hits were identified as involved in protein secretion, of which 51 were novel hits not previously associated with protein secretion. Many of the hits identified also resulted in a phenotype of altered Golgi morphology, and five of these were investigated further. Two novel hits that localised to the Golgi were identified, TMEM220 and GPR161. GPR161 likely mediates its effect on Golgi morphology by its interaction with golgin A5. This thesis presents new insights into both conventional and unconventional protein secretion. Insights into the mechanism for galaectin-3 secretion were revealed. A novel Golgi-localised regulator of secretion, GRP161, was identified, with a mechanism for action suggested. Importantly, the list of 51 novel genes identified will serve as a useful resource for other researchers investigating protein secretion.

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The FlbA-regulated predicted transcription factor rpnR of Aspergillus niger is involved in protein secretion and stress resistance

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