Exploiting secretory pathways for production of heterologous proteins is highly advantageous with respect to efficient downstream processing. In eukaryotic systems the vast majority of heterologous proteins for biotechnological application is exported via the canonical endoplasmic reticulum–Golgi pathway. In the endomembrane system target proteins are often glycosylated and may thus be modified with foreign glycan patterns. This can be destructive for their activity or cause immune reactions against therapeutic proteins. Hence, using unconventional secretion for protein expression is an attractive alternative. In the fungal model Ustilago maydis, chitinase Cts1 is secreted via an unconventional pathway connected to cell separation which can be used to co-export heterologous proteins. Here, we apply this mechanism for the production of nanobodies. First, we achieved expression and unconventional secretion of a functional nanobody directed against green fluorescent protein (Gfp). Second, we found that Cts1 binds to chitin and that this feature can be applied to generate a Gfp-trap. Thus, we demonstrated the dual use of Cts1 serving both as export vehicle and as purification tag. Finally, we established and optimized the production of a nanobody against botulinum toxin A and hence describe the first pharmaceutically relevant target exported by Cts1-mediated unconventional secretion.
mRNA transport determines spatiotemporal protein expression. Transport units are higher‐order ribonucleoprotein complexes containing cargo mRNAs, RNA‐binding proteins and accessory proteins. Endosomal mRNA transport in fungal hyphae belongs to the best‐studied translocation mechanisms. Although several factors are known, additional core components are missing. Here, we describe the 232 kDa protein Upa2 containing multiple PAM2 motifs (poly[A]‐binding protein [Pab1]‐associated motif 2) as a novel core component. Loss of Upa2 disturbs transport of cargo mRNAs and associated Pab1. Upa2 is present on almost all transport endosomes in an mRNA‐dependent manner. Surprisingly, all four PAM2 motifs are dispensable for function during unipolar hyphal growth. Instead, Upa2 harbours a novel N‐terminal effector domain as important functional determinant as well as a C‐terminal GWW motif for specific endosomal localisation. In essence, Upa2 meets all the criteria of a novel core component of endosomal mRNA transport and appears to carry out crucial scaffolding functions.
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