Maturation of dense core granules in wild type and mutant Tetrahymena thermophila.

Turkewitz, Aaron P.; Madeddu, Luisa; Kelly, Regis B.

doi:10.1002/j.1460-2075.1991.tb07727.x

Cited by 66 publications

(80 citation statements)

References 42 publications

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“…Grt1p was first detected as one of the most abundant DCG components released during exocytosis (32). Biochemical analysis showed that Grt1p differs in its solubility from the Grl proteins and also that it is packaged intact in DCGs rather than undergoing proteolytic processing (31).…”

mentioning

confidence: 99%

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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confidence: 99%

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

2009

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“…Cell Calcium (1998) 23 (5), [349][350][351][352][353][354][355][356][357][358][359][360] genetic analyses [55,56]. This is substantiated by our present finding of an abnormal set of trichynins in tnd1, undergoing only partial DTT-induced MW shift on SDS-PAGE, and greatly reduced Caz+-binding.…”

Section: Lack Of Contents' Decondensation In Tnd1 Cellsmentioning

confidence: 57%

“…Note that equal amounts of protein were applied to lanes in Figure 11. Imperfect cleavage can be only one reason of reduced 45Ca binding, since the 45 kDa band binds no [55,56].…”

Section: Experiments With Isolated Trichocysts Sds-page and 45ca Ovementioning

confidence: 99%

An exocytotic mutant of Paramecium caudatum: membrane fusion without secretory contents release

et al. 1998

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Summary This is a detailed characterization of a secretory mutant incapable of releasing secretory contents despite normal exocytotic membrane fusion performance. Trichocyst non-discharge strain tnd1 of Paramecium caudatum and its wildtype (wt) both show a transient cortical [Ca 2 +l; increase and exocytotic membrane fusion in response to the polyamine secretagogue, aminoethyldextran (AEO), or to caffeine, tnd1 cells frequently display spontaneous Ca 2 + signals parallelled by spontaneous exocytotic membrane fusion. This remains undetected, unless the trichocyst matrix is shown to be freely accessible to the inert, non-membrane permeable fluorochrome, F 2 FITC, from the outside. In these tnd1 cells, spontaneous and AEO-or caffeine-induced membrane fusion, always without contents expulsion by decondensation (i.e. several-fold stretching), is ascertained by electron microscopy. Exocytotic openings, with condensed trichocysts retained, may persist for hours without impairing cells. Trichocyst decondensation normally requires micromolar [Ca 2 +]e' but an increase to 10 mM has no effect on tnd1 trichocyst expansion in vivo or in vitro (when isolated and exposed to ionophore A23187 + Ca 2 +). Paracrystalline packing of the major secretory components (trichynins) does occur, despite incomplete proteolytic precursor processing (according to SOS-PAGE). However, 45Ca 2 +-binding by secretory components is considerably reduced -the likely cause of the non-discharge phenotype. Our findings imply significant untriggered membrane fusion in a system normally following the triggered pathway and clear separation of exocytotic membrane fusion from any later Ca 2 +-dependent steps of the secretory cycle.

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“…Similar mutants defective on different levels of the secretory pathway have been obtained from Tetrahymena (Sauer and Kelly, 1995;Melia et al, 1998). With these cells it was also possible to show that secretory content sorting precedes condensation into a mature form (Turkewitz et al, 1991;Bowman and Turkewitz, 2001).…”

Section: Ill Standard Route: Endoplasmic Reticulum To Golgi Complex mentioning

confidence: 99%

Molecular Aspects of Membrane Trafficking in Paramecium

Plattner

Kissmehl

2003

International Review of Cytology

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Results achieved in tile molecular biology of Paramecium have shed new light on its elaborate membrane trafficking system. Paramecium disposes not only of the standard routes (endoplasmic reticulum ---+ Golgi ---+ Iysosomes or secretory vesicles; endo-and phagosomes ---+ Iysosomes/digesting vacuoles), but also of some unique features, e.g. and elaborate phagocytic route with the cytoproct and membrane recycling to the cytopharynx, as well as the osmoregulatory system with multiple membrane fusion sites. Exocytosis sites for trichocysts (dense-core secretory vesicles), parasomal sacs (coated pits), and terminal cisternae (early endosomes) display additional regularly arranged predetermined fusion/fission sites, which now can be discussed on a molecular basis. Considering the regular, repetitive arrangements of membrane components, availability of mutants for complementation studies, sensitivity to gene silencing, and so on, Paramecium continues to be a valuable model system for analyzing membrane interactions. This review intends to set a new baseline for ongoing work along these lines.

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Maturation of dense core granules in wild type and mutant Tetrahymena thermophila.

Cited by 66 publications

References 42 publications

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

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

An exocytotic mutant of Paramecium caudatum: membrane fusion without secretory contents release

Molecular Aspects of Membrane Trafficking in Paramecium

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