Genomic and Proteomic Evidence for a Second Family of Dense Core Granule Cargo Proteins in <i>Tetrahymena thermophila</i>

Bowman, Grant R.; Smith, Daryl G.S.; Siu, Kin Wai Michael; Pearlman, Ronald E.; Turkewitz, Aaron P.

doi:10.1111/j.1550-7408.2005.00045.x

Cited by 29 publications

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“…Third, the utility of the system was recently augmented by the complete sequencing and preliminary annotation of the macronuclear genome (22,73). The availability of this information has allowed proteomic analyses of isolated organelles, using tandem mass spectrometry (10,71). In this study, we purified phagosomes from T. thermophila and characterized their protein composition by multidimensional separation coupled with tandem mass spectrometry (71,86).…”

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The Tetrahymena thermophila Phagosome Proteome

et al. 2006

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In vertebrates, phagocytosis occurs mainly in specialized cells of the immune system and serves as a primary defense against invading pathogens, but it also plays a role in clearing apoptotic cells and in tissue remodeling during development. In contrast, unicellular eukaryotes, such as the ciliate Tetrahymena thermophila, employ phagocytosis to ingest and degrade other microorganisms to meet their nutritional needs. To learn more about the protein components of the multistep process of phagocytosis, we carried out an analysis of the Tetrahymena phagosome proteome. Tetrahymena cells were fed polystyrene beads, which allowed for the efficient purification of phagosomes. The protein composition of purified phagosomes was then analyzed by multidimensional separation coupled with tandem mass spectrometry. A total of 453 peptides were identified that resulted in the identification of 73 putative phagosome proteins. Twenty-eight of the proteins have been implicated in phagocytosis in other organisms, indicating that key aspects of phagocytosis were conserved during evolution. Other identified proteins have not previously been associated with phagocytosis, including some of unknown function. Live-cell confocal fluorescence imaging of Tetrahymena strains expressing green fluorescent protein-tagged versions of four of the identified phagosome proteins provided evidence that at least three of the proteins (including two with unknown functions) are associated with phagosomes, indicating that the bulk of the proteins identified in the analyses are indeed phagosome associated.

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The Tetrahymena thermophila Phagosome Proteome

et al. 2006

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“…In addition to the GRL family-encoded proteins, 13 other lumenal DCG proteins have been putatively or definitively identified in Tetrahymena, and homologous proteins are predicted in the Paramecium genome (6). The entire set belongs to a gene family that is defined by a carboxy-terminal ␤/␥-crystallin domain, which may function as a DCG-targeting motif (16).…”

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Independent Transport and Sorting of Functionally Distinct Protein Families in Tetrahymena thermophila Dense Core Secretory Granules

2009

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Dense core granules (DCGs) in Tetrahymena thermophila contain two protein classes. Proteins in the first class, called granule lattice (Grl), coassemble to form a crystalline lattice within the granule lumen. Lattice expansion acts as a propulsive mechanism during DCG release, and Grl proteins are essential for efficient exocytosis. The second protein class, defined by a C-terminal ␤/␥-crystallin domain, is poorly understood. Here, we have analyzed the function and sorting of Grt1p (granule tip), which was previously identified as an abundant protein in this family. Cells lacking all copies of GRT1, together with the closely related GRT2, accumulate wild-type levels of docked DCGs. Unlike cells disrupted in any of the major GRL genes, ⌬GRT1 ⌬GRT2 cells show no defect in secretion, indicating that neither exocytic fusion nor core expansion depends on GRT1. These results suggest that Grl protein sorting to DCGs is independent of Grt proteins. Consistent with this, the granule core lattice in ⌬GRT1 ⌬GRT2 cells appears identical to that in wild-type cells by electron microscopy, and the only biochemical component visibly absent is Grt1p itself. Moreover, gel filtration showed that Grl and Grt proteins in cell homogenates exist in nonoverlapping complexes, and affinity-isolated Grt1p complexes do not contain Grl proteins. These data demonstrate that two major classes of proteins in Tetrahymena DCGs are likely to be independently transported during DCG biosynthesis and play distinct roles in granule function. The role of Grt1p may primarily be postexocytic; consistent with this idea, DCG contents from ⌬GRT1 ⌬GRT2 cells appear less adhesive than those from the wild type.

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“…The most abundant proteins stored in and released from Tetrahymena mucocysts belong to two families, called GRT (for granule tip) and GRL (for granule lattice) (22,23). Studies of the Grl proteins, and the related P. tetraurelia tmp proteins, have demonstrated that extensive proteolytic processing occurs during mucocyst and trichocyst synthesis (20,(24)(25)(26).…”

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“…During exocytosis, the secretory proteins are rapidly extruded, due to the fact that the crystalline lattices expand rapidly and directionally. Thus, the function of secretory organelles in ciliates depends upon the assembly of protein crystals that can undergo spring-like elongation (20,21).The most abundant proteins stored in and released from Tetrahymena mucocysts belong to two families, called GRT (for granule tip) and GRL (for granule lattice) (22,23). Studies of the Grl proteins, and the related P. tetraurelia tmp proteins, have demonstrated that extensive proteolytic processing occurs during mucocyst and trichocyst synthesis (20,(24)(25)(26).…”

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Secretion of Polypeptide Crystals from Tetrahymena thermophila Secretory Organelles (Mucocysts) Depends on Processing by a Cysteine Cathepsin, Cth4p

2015

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In many organisms, sophisticated mechanisms facilitate release of peptides in response to extracellular stimuli. In the ciliate Tetrahymena thermophila, efficient peptide secretion depends on specialized vesicles called mucocysts that contain dense crystalline cores that expand rapidly during exocytosis. Core assembly depends of endoproteolytic cleavage of mucocyst proproteins by an aspartyl protease, cathepsin 3 (CTH3). Here, we show that a second enzyme identified by expression profiling, Cth4p, is also required for processing of proGrl proteins and for assembly of functional mucocysts. Cth4p is a cysteine cathepsin that localizes partially to endolysosomal structures and appears to act downstream of, and may be activated by, Cth3p. Disruption of CTH4 results in cells (⌬cth4) that show aberrant trimming of Grl proproteins, as well as grossly aberrant mucocyst exocytosis. Surprisingly, ⌬cth4 cells succeed in assembling crystalline mucocyst cores. However, those cores do not undergo normal directional expansion during exocytosis, and they thus fail to efficiently extrude from the cells. We could phenocopy the ⌬cth4 defects by mutating conserved catalytic residues, indicating that the in vivo function of Cth4p is enzymatic. Our results indicate that as for canonical proteins packaged in animal secretory granules, the maturation of mucocyst proproteins involves sequential processing steps. The ⌬cth4 defects uncouple, in an unanticipated way, the assembly of mucocyst cores and their subsequent expansion and thereby reveal a previously unsuspected aspect of polypeptide secretion in ciliates. R egulated secretion of peptides from intracellular stores plays many key roles in intercellular communication and tissue coordination in animals (1). A highly specialized organelle in neuroendocrine cells, the secretory granule, is required for peptide generation, storage, and release (2). The bioactive peptides are generated during secretory granule formation by the action of proteolytic enzymes on polypeptide precursors (3). Proteolytic processing occurs in a post-trans-Golgi network (TGN) compartment, the immature granule, to which propeptides are sorted away from bulk protein traffic, and is a multistep process involving several classes of enzymes (4-6). The best studied among these are a family of serine proteases called prohormone convertases (7). Related to bacterial subtilisins, prohormone convertases (PCs) catalyze endoproteolytic cleavage at defined motifs in their substrates. The products of that cleavage can then undergo trimming, for which one important factor is carboxypeptidase E (CPE) (8). Defects in both PCs and CPE are linked with disease (9, 10).Signaling via peptide secretion appears to be widespread in eukaryotes and has also been well documented for fungi, amoebozoa, and plants(11-13). In the oligohymenophorean ciliates Tetrahymena thermophila and Paramecium tetraurelia, regulated peptide secretion occurs from organelles that share striking similarities with secretory granules in animals (14, 15), although...

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Genomic and Proteomic Evidence for a Second Family of Dense Core Granule Cargo Proteins in Tetrahymena thermophila

Cited by 29 publications

References 31 publications

The Tetrahymena thermophila Phagosome Proteome

The Tetrahymena thermophila Phagosome Proteome

Independent Transport and Sorting of Functionally Distinct Protein Families in Tetrahymena thermophila Dense Core Secretory Granules

Secretion of Polypeptide Crystals from Tetrahymena thermophila Secretory Organelles (Mucocysts) Depends on Processing by a Cysteine Cathepsin, Cth4p

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