Remodeling the Specificity of an Endosomal CORVET Tether Underlies Formation of Regulated Secretory Vesicles in the Ciliate Tetrahymena thermophila

Abstract

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, Δvps8a 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 that 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.

Diversification of CORVET tethers facilitates transport complexity inTetrahymena thermophila

“…T. thermophila expresses two Vps33, two Vps16, two Vps3, four Vps18 and six Vps8 paralogs. We previously discovered, starting from a forward genetic approach, that the "a" paralog of Vps8 was essential for mucocyst formation (Sparvoli et al, 2018). In particular, ∆vps8a cells failed to deliver a subset of mucocyst cargo proteins, consistent with the idea that a specific pathway depended upon the activity of a Vps8a-containing tether.…”

“…S2A). VPS8C and D are essential genes (Sparvoli et al, 2018). Since the cells relying on mNeontagged Vps8c and 8d had no detectible growth phenotypes, we infer that tagging does not interfere with protein activity.…”

“…Tetrahymena and related ciliates possess an atypical complement of genes encoding endolysosomal tethers and other trafficking genes, indicating a significant bottleneck in ancestors of this lineage that resulted in secondary losses, and particularly in Rab GTPases (Sparvoli et al, 2018). Most relevant here is that both HOPS-specific subunits were lost, but most other subunits (i.e., the core subunits, as well as the CORVET-specific subunits) were retained and expanded into multiple paralogs (Klinger et al, 2013).…”

“…Moreover, these paralogs have been maintained in multiple species suggesting that they provide important, non-redundant functions. The six VPS8 paralogs in T. thermophila differ in their transcriptional profiles, consistent with functional diversification (Sparvoli et al, 2018). To determine if each Vps8 paralog belongs to a unique biochemical complex, we expressed each as an endogenously-FLAG-tagged fusion.…”

“…Vps8a is one of six Vps8 paralogs expressed in Tetrahymena. These paralogs are ancient in origin: the split between even the most-closely related pair (VPS8A and VPS8C) predates the ~22MYA divergence between T. thermophila and T. malaccensis (Sparvoli et al, 2018). Moreover, these paralogs have been maintained in multiple species suggesting that they provide important, non-redundant functions.…”