A Reporter System to Study Unconventional Secretion of Proteins Avoiding N-Glycosylation in Ustilago maydis

Stock, Janpeter; Terfrüchte, Marius; Schipper, Kerstin

doi:10.1007/978-1-4939-3804-9_10

Cited by 13 publications

(19 citation statements)

References 21 publications

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“…The supernatant was transferred to a new tube and stored on 4°C. The cell pellet was used to prepare native cell extracts used for the Cts1, Gus and LacZ assay (modified from Stock et al, 2016 ). To this end the cell pellet was resuspended in 2 ml ice-cold native extraction buffer (1 mM phenylmethylsulfonylfluorid (PMSF), 2.5 mM benzamidine hydrochloride hydrate, 1 μM pepstatin; 100 μL Roche EDTA-free protease inhibitor cocktail 50×; dissolve in PBS pH 7.4).…”

Section: Methodsmentioning

confidence: 99%

A Novel Factor Essential for Unconventional Secretion of Chitinase Cts1

et al. 2020

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Subcellular targeting of proteins is essential to orchestrate cytokinesis in eukaryotic cells. During cell division of Ustilago maydis , for example, chitinases must be specifically targeted to the fragmentation zone at the site of cell division to degrade remnant chitin and thus separate mother and daughter cells. Chitinase Cts1 is exported to this location via an unconventional secretion pathway putatively operating in a lock-type manner. The underlying mechanism is largely unexplored. Here, we applied a forward genetic screen based on UV mutagenesis to identify components essential for Cts1 export. The screen revealed a novel factor termed Jps1 lacking known protein domains. Deletion of the corresponding gene confirmed its essential role for Cts1 secretion. Localization studies demonstrated that Jps1 colocalizes with Cts1 in the fragmentation zone of dividing yeast cells. While loss of Jps1 leads to exclusion of Cts1 from the fragmentation zone and strongly reduced unconventional secretion, deletion of the chitinase does not disturb Jps1 localization. Yeast-two hybrid experiments indicate that the two proteins might interact. In essence, we identified a novel component of unconventional secretion that functions in the fragmentation zone to enable export of Cts1. We hypothesize that Jps1 acts as an anchoring factor for Cts1.

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Section: Methodsmentioning

confidence: 99%

A Novel Factor Essential for Unconventional Secretion of Chitinase Cts1

et al. 2020

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“…The preparation of cell extracts and precipitation of proteins from cell-free culture supernatants have been described before [24,28]. Purification was performed according to the Qiagen Expressionist manual.…”

Section: Methodsmentioning

confidence: 99%

Applying Unconventional Secretion in Ustilago maydis for the Export of Functional Nanobodies

Terfrüchte

Reindl

Jankowski

et al. 2017

IJMS

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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.

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“…Therefore, these data support the view that it is possible for glycosylated proteins to be exported through unconventional pathways. On the other hand, Stock et al (2016) could show, through heterologous expression of a modified b-glucuronidase (GUS) reported gene in Ustilago maydis, that glycosylation only took place when the ER secretory pathway directed protein secretion. No glycosylation was observed in the UPS pathway.…”

Section: Secretion Of Proteins and Peptides By Plants And Fungimentioning

confidence: 99%

The Multiple Facets of Plant–Fungal Interactions Revealed Through Plant and Fungal Secretomics

2020

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The plant secretome is usually considered in the frame of proteomics, aiming at characterizing extracellular proteins, their biological roles and the mechanisms accounting for their secretion in the extracellular space. In this review, we aim to highlight recent results pertaining to secretion through the conventional and unconventional protein secretion pathways notably those involving plant exosomes or extracellular vesicles. Furthermore, plants are well known to actively secrete a large array of different molecules from polymers (e.g. extracellular RNA and DNA) to small compounds (e.g. ATP, phytochemicals, secondary metabolites, phytohormones). All of these play pivotal roles in plant-fungi (or oomycetes) interactions, both for beneficial (mycorrhizal fungi) and deleterious outcomes (pathogens) for the plant. For instance, recent work reveals that such secretion of small molecules by roots is of paramount importance to sculpt the rhizospheric microbiota. Our aim in this review is to extend the definition of the plant and fungal secretomes to a broader sense to better understand the functioning of the plant/microorganisms holobiont. Fundamental perspectives will be brought to light along with the novel tools that should support establishing an environment-friendly and sustainable agriculture.

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A Reporter System to Study Unconventional Secretion of Proteins Avoiding N-Glycosylation in Ustilago maydis

Cited by 13 publications

References 21 publications

A Novel Factor Essential for Unconventional Secretion of Chitinase Cts1

A Novel Factor Essential for Unconventional Secretion of Chitinase Cts1

Applying Unconventional Secretion in Ustilago maydis for the Export of Functional Nanobodies

The Multiple Facets of Plant–Fungal Interactions Revealed Through Plant and Fungal Secretomics

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