Turkewitz, A. P. | Search bioRxiv

bioRxiv2017

An evolutionary switch in the specificity of an endosomal CORVET tether underlies formation of regulated secretory vesicles in the ciliate Tetrahymena thermophila

In the endocytic pathway of animals, two related complexes, called CORVET (Class C Core Vacuole/Endosome Transport) and HOPS (Homotypic fusion and protein sorting), act as both tethers and fusion factors for early and late endosomes, respectively. Mutations in CORVET or HOPS lead to trafficking defects and contribute to human disease including immune dysfunction. HOPS and CORVET are conserved throughout eukaryotes but remarkably, in the ciliate Tetrahymena thermophila, the HOPS-specific subunits are absent while CORVET-specific subunits have proliferated. VPS8 (Vacuolar Protein Sorting), a CORVET subunit, expanded to 6 paralogs in Tetrahymena. This expansion correlated with loss of HOPS within a ciliate subgroup including the Oligohymenophorea, which contains Tetrahymena. As uncovered via forward genetics, a single VPS8 paralog in Tetrahymena (VPS8A) is required to synthesize prominent secretory granules called mucocysts. More specifically,{triangleup} vps8a cells fail to deliver a subset of cargo proteins to developing mucocysts, instead accumulating that cargo in vesicles also bearing the mucocyst sorting receptor, Sor4p. Surprisingly, although this transport step relies on CORVET, it does not appear to involve early endosomes. Instead, Vps8a associates with the late endosomal/lysosomal marker Rab7, indicating target specificity switching occurred in CORVET subunits during the evolution of ciliates. Mucocysts belong to a markedly diverse and understudied class of protist secretory organelles called extrusomes. Our results underscore that biogenesis of mucocysts depends on endolysosomal trafficking, revealing parallels with invasive organelles in apicomplexan parasites and suggesting that a wide array of secretory adaptations in protists, like in animals, depend on mechanisms related to lysosome biogenesis.\n\nAbbreviations

cell biology↗

bioRxiv2017

The Co-regulation Data Harvester for Tetrahymenathermophila: automated high-throughput geneannotation and functional inference in a microbialeukaryote

Identifying co-regulated genes can provide a useful approach for defining pathway-specific machinery in an organism. To be efficient, this approach relies on thorough genome annotation, which is not available for most organisms with sequenced genomes. Studies in Tetrahymena thermophila, the most experimentally accessible ciliate, have generated a rich transcriptomic database covering many well-defined physiological states. Genes that are involved in the same pathway show significant co-regulation, and screens based on gene co-regulation have identified novel factors in specific pathways, for example in membrane trafficking. However, a limitation has been the relatively sparse annotation of the Tetrahymena genome, making it impractical to approach genome-wide analyses. We have therefore developed an efficient approach to analyze both co-regulation and gene annotation, called the Co-regulation Data Harvester (CDH). The CDH automates identification of co-regulated genes by accessing the Tetrahymena transcriptome database, determines their orthologs in other organisms via reciprocal BLAST searches, and collates the annotations of those orthologs' functions. Inferences drawn from the CDH reproduce and expand upon experimental findings in Tetrahymena. The CDH, which is freely available, represents a powerful new tool for analyzing cell biological pathways in Tetrahymena. Moreover, to the extent that genes and pathways are conserved between organisms, the inferences obtained via the CDH should be relevant, and can be explored, in many other systems.

bioinformatics↗