Abstract The formation of dense core granules (DCGs) requires both the sorting of granule contents from other secretory proteins and a postsorting maturation process. The Tetrahymena thermophila strain SB281 fails to synthesize DCGs, and previous analysis suggested that the defect lay at or near the sorting step. Because this strain represents one of the very few mutants in this pathway, we have undertaken a more complete study of the phenotype. Genetic epistasis analysis places the defect upstream of those in two other characterized Tetrahymena mutants. Using immunofluorescent detection of granule content proteins, as well as GFP tagging, we describe a novel cytoplasmic compartment to which granule contents can be sorted in growing SB281 cells. Cell fusion experiments indicate that this compartment is not a biosynthetic intermediate in DCG synthesis. Sorting in SB281 is strongly conditional with respect to growth. When cells are starved, the storage compartment is degraded and de novo synthesized granule proteins are rapidly secreted. The mutation in SB281 therefore appears to affect DCG synthesis at the level of both sorting and maturation.
Significance Bacteria use molecular partitioning systems based on the ATPase ParA to segregate chromosome centromeres before cell division, but how these machines target centromeres to specific locations is unclear. This study shows that, in Caulobacter crescentus , a multimeric complex composed of the PopZ protein directs the ParA machine to transfer centromeres to the cell pole. Spent ParA subunits released from the mitotic apparatus during segregation are recruited throughout a 3D PopZ matrix at the pole. ParA recruitment and sequestration by PopZ stimulates the cell-pole proximal recycling of ParA into a nucleoid-bound complex to ensure pole-specific centromere transfer. PopZ therefore utilizes a 3D scaffolding strategy to create a subcellular microdomain that directly regulates the function of the bacterial centromere segregation machine.
Summary 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, ∆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. Abbreviations LRO (Lysosome-related organelle) HOPS (homotypic fusion and protein sorting complex) CORVET (Class C core Vacuole/Endosome Transport) VPS (vacuolar protein sorting) GRL (granule lattice) GRT (granule tip) Igr (Induced upon granule regeneration) SNARE (Soluble NSF attachment protein receptor) LECA (last eukaryotic common ancestor)
Significance Despite being the simplest organisms, bacteria have complex subcellular anatomies. How does such organization occur in openly diffusive cytoplasm? We find that the cell pole organizing protein PopZ facilitates network formation by binding directly to at least eight other proteins. The binding region in PopZ is intrinsically disordered, suggesting PopZ has a flexible structure that adopts a different interface for each partner protein. In this way, PopZ resembles eukaryotic hub proteins, such as p53 and BRCA1, which coordinate complex signaling networks. Rapid cycles of binding, unbinding, and rebinding within PopZ networks indicate that bacterial cell poles, similar to their eukaryotic counterparts, are highly dynamic structures.
The ubiquitous bacterium Caulobacter crescentus holds promise to be used in bioremediation applications due to its ability to mineralize U(VI) under aerobic conditions. Here, cell free extracts of C. crescentus grown in the presence of uranyl nitrate [U(VI)], potassium chromate [Cr(VI)], or cadmium sulfate [Cd(II)] were used for label-free proteomic analysis. Proteins involved in two-component signaling and amino acid metabolism were up-regulated in response to all three metals, and proteins involved in aerobic oxidative phosphorylation and chemotaxis were down-regulated under these conditions. Clustering analysis of proteomic enrichment revealed that the three metals also induce distinct patterns of up- or down-regulated expression among different functional classes of proteins. Under U(VI) exposure, a phytase enzyme and an ABC transporter were up-regulated. Heat shock and outer membrane responses were found associated with Cr(VI), while efflux pumps and oxidative stress proteins were up-regulated with Cd(II). Experimental validations were performed on select proteins. We found that a phytase plays a role in U(VI) and Cr(VI) resistance and detoxification and that a Cd(II)-specific transporter confers Cd(II) resistance. Interestingly, analysis of promoter regions in genes associated with differentially expressed proteins suggests that U(VI) exposure affects cell cycle progression.
