Abstract
BackgroundA highly regulated trafficking of cargo vesicles in eukaryotes performs protein delivery to a variety of cellular compartments of endomembrane system. The two main routes, the secretory and the endocytic pathways have pivotal functions in uni- and multi-cellular organisms. Protein delivery and targeting includes cargo recognition, vesicle formation and fusion. Developing new tools to modulate protein trafficking allows better understanding the endomembrane system mechanisms and their regulation. The compound Sortin2 has been described as a protein trafficking modulator affecting targeting of the vacuolar protein carboxypeptidase Y (CPY), triggering its secretion in Saccharomyces cerevisiae.ResultsA reverse chemical-genetics approach was used to identify key proteins for Sortin2 bioactivity. A genome-wide Sortin2 resistance screen revealed six yeast deletion mutants that do not secrete CPY when grown at Sortin2 condition where the parental strain does: met18, sla1, clc1, dfg10, dpl1 and yjl175w. Integrating mutant phenotype and gene ontology annotation of the corresponding genes and their interactome pointed towards a high representation of genes involved in the endocytic process. In wild type yeast endocytosis towards the vacuole was faster in presence of Sortin2, which further validates the data of the genome-wide screen. This effect of Sortin2 depends on structural features of the molecule, suggesting compound specificity. Sortin2 did not affect endocytic trafficking in Sortin2-resistant mutants, strongly suggesting that the Sortin2 effects on the secretory and endocytic pathways are linked.ConclusionsOverall, the results reveal that Sortin2 enhances the endocytic transport pathway in Saccharomyces cerevisiae. This cellular effect is most likely at the level where secretory and endocytic pathways are merged. Them Sortin2 specificity over the endomembrane system places it as a powerful biological modulator for cell biology.Electronic supplementary materialThe online version of this article (doi:10.1186/s40659-015-0032-9) contains supplementary material, which is available to authorized users.
Highlights
A highly regulated trafficking of cargo vesicles in eukaryotes performs protein delivery to a variety of cellular compartments of endomembrane system
It was anticipated that this resistance screening would identify proteins required for Sortin2 bioactivity that were not codified by an essential gene
The results showed that sla1, met18 and clc1 were resistant to up to 40 μM Sortin2
Summary
A highly regulated trafficking of cargo vesicles in eukaryotes performs protein delivery to a variety of cellular compartments of endomembrane system. Eukaryotes have developed an intricate system of protein delivery to various cellular compartments, based on highly regulated trafficking of cargo vesicles. Vesicle formation and fusion represent the core of endomembrane system processes and form two main routes, the secretory and the endocytic pathways. These two pathways have pivotal functions in uni- and multicellular organisms. Chemical genomics is a powerful tool to discover such new biomodulators It employs large diverse collections of compounds to identify small bioactive molecules in order to manipulate biological pathways in a similar fashion to classical genetics [3, 4].
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