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

2009 ◽  
Vol 8 (10) ◽  
pp. 1575-1583 ◽  
Author(s):  
Abdur Rahaman ◽  
Wei Miao ◽  
Aaron P. Turkewitz
Keyword(s):  
Tetrahymena Thermophila ◽  
Secretory Granules ◽  
Gel Filtration ◽  
Dense Core ◽  
Crystalline Lattice ◽  
Wild Type ◽  
Biochemical Component ◽  
Protein Classes ◽  
Dense Core Granules

ABSTRACT 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 β/γ-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, ΔGRT1 ΔGRT2 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 ΔGRT1 ΔGRT2 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 ΔGRT1 ΔGRT2 cells appear less adhesive than those from the wild type.

scholarly journals Granule lattice protein 1 (Grl1p), an acidic, calcium-binding protein in Tetrahymena thermophila dense-core secretory granules, influences granule size, shape, content organization, and release but not protein sorting or condensation.

1996 ◽  
Vol 135 (6) ◽  
pp. 1775-1787 ◽  
Author(s):  
N D Chilcoat ◽  
S M Melia ◽  
A Haddad ◽  
A P Turkewitz
Keyword(s):  
Calcium Binding ◽  
Plasma Membranes ◽  
Tetrahymena Thermophila ◽  
Secretory Granules ◽  
Protein Sorting ◽  
Dense Core ◽  
Rapid Expansion ◽  
Crystalline Lattice ◽  
Wild Type ◽  
Granule Proteins

The electron-dense cores of regulated secretory granules in the ciliate Tetrahymena thermophila are crystal lattices composed of multiple proteins. Granule synthesis involves a series of steps beginning with protein sorting, followed by the condensation and precise geometric assembly of the granule cargo. These steps may to various degrees be determined by the cargo proteins themselves. A prominent group of granule proteins, in ciliates as well as in vertebrate neuronal and endocrine cells, are acidic, heat-stable, and bind calcium. We focused on a protein with these characteristics named granule lattice protein 1 (Grl1p), which represents 16% of total granule contents, and we have now cloned the corresponding gene. Mutants in which the macronuclear copies of GRL1 have been disrupted continue to synthesize dense-core granules but are nonetheless defective in regulated protein secretion. To understand the nature of this defect, we characterized mutant and wild-type granules. In the absence of Grl1p, the sorting of the remaining granule proteins appears normal, and they condense to form a well-defined core. However, the condensed cores do not demonstrate a visible crystalline lattice, and are notably different from wild type in size and shape. The cellular secretion defect arises from failure of the aberrant granule cores to undergo rapid expansion and extrusion after exocytic fusion of the granule and plasma membranes. The results suggest that sorting, condensation, and precise granule assembly are distinct in their requirements for Grl1p.

Analysis of a Mutant Exhibiting Conditional Sorting to Dense Core Secretory Granules in Tetrahymena thermophila

Genetics ◽  
2001 ◽  
Vol 159 (4) ◽  
pp. 1605-1616
Author(s):  
Grant R Bowman ◽  
Aaron P Turkewitz
Keyword(s):  
De Novo ◽  
Tetrahymena Thermophila ◽  
Previous Analysis ◽  
Secretory Granules ◽  
Dense Core ◽  
Secretory Proteins ◽  
Epistasis Analysis ◽  
Complete Study ◽  
Genetic Epistasis ◽  
Dense Core Granules

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.

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

1991 ◽  
Vol 10 (8) ◽  
pp. 1979-1987 ◽  
Author(s):  
A.P. Turkewitz ◽  
L. Madeddu ◽  
R.B. Kelly
Keyword(s):  
Tetrahymena Thermophila ◽  
Dense Core ◽  
Wild Type ◽  
Dense Core Granules

scholarly journals New Class of Cargo Protein in Tetrahymena thermophila Dense Core Secretory Granules

2002 ◽  
Vol 1 (4) ◽  
pp. 583-593 ◽  
Author(s):  
Alex Haddad ◽  
Grant R. Bowman ◽  
Aaron P. Turkewitz
Keyword(s):  
Tetrahymena Thermophila ◽  
Secretory Granules ◽  
Dense Core ◽  
Biologically Active ◽  
Paramecium Tetraurelia ◽  
New Class ◽  
Cargo Protein ◽  
Before And After ◽  
Granule Proteins ◽  
Dense Core Granules

ABSTRACT Regulated exocytosis of dense core secretory granules releases biologically active proteins in a stimulus-dependent fashion. The packaging of the cargo within newly forming granules involves a transition: soluble polypeptides condense to form water-insoluble aggregates that constitute the granule cores. Following exocytosis, the cores generally disassemble to diffuse in the cell environment. The ciliates Tetrahymena thermophila and Paramecium tetraurelia have been advanced as genetically manipulatable systems for studying exocytosis via dense core granules. However, all of the known granule proteins in these organisms condense to form the architectural units of lattices that are insoluble both before and after exocytosis. Using an approach designed to detect new granule proteins, we have now identified Igr1p (induced during granule regeneration). By structural criteria, it is unrelated to the previously characterized lattice-forming proteins. It is distinct in that it is capable of dissociating from the insoluble lattice following secretion and therefore represents the first diffusible protein identified in ciliate granules.

scholarly journals An aspartyl cathepsin, CTH3, is essential for proprotein processing during secretory granule maturation in Tetrahymena thermophila

2014 ◽  
Vol 25 (16) ◽  
pp. 2444-2460 ◽  
Author(s):  
Santosh Kumar ◽  
Joseph S. Briguglio ◽  
Aaron P. Turkewitz
Keyword(s):  
Direct Evidence ◽  
Fluorescent Protein ◽  
Tetrahymena Thermophila ◽  
Secretory Granules ◽  
Processing Enzymes ◽  
Proprotein Processing ◽  
Type Gene ◽  
Granule Maturation ◽  
Dense Core Granules

In Tetrahymena thermophila, peptides secreted via dense-core granules, called mucocysts, are generated by proprotein processing. We used expression profiling to identify candidate processing enzymes, which localized as cyan fluorescent protein fusions to mucocysts. Of note, the aspartyl cathepsin Cth3p plays a key role in mucocyst-based secretion, since knockdown of this gene blocked proteolytic maturation of the entire set of mucocyst proproteins and dramatically reduced mucocyst accumulation. The activity of Cth3p was eliminated by mutation of two predicted active-site mutations, and overexpression of the wild-type gene, but not the catalytic-site mutant, partially rescued a Mendelian mutant defective in mucocyst proprotein processing. Our results provide the first direct evidence for the role of proprotein processing in this system. Of interest, both localization and the CTH3 disruption phenotype suggest that the enzyme provides non–mucocyst-related functions. Phylogenetic analysis of the T. thermophila cathepsins, combined with prior work on the role of sortilin receptors in mucocyst biogenesis, suggests that repurposing of lysosomal enzymes was an important step in the evolution of secretory granules in ciliates.

scholarly journals Role of Arg-401 of cytosolic serine hydroxymethyltransferase in subunit assembly and interaction with the substrate carboxy group

1997 ◽  
Vol 327 (3) ◽  
pp. 877-882 ◽  
Author(s):  
Junutula Reddy JAGATH ◽  
Naropantul APPAJI RAO ◽  
Handanahal SubbaRao SAVITHRI
Keyword(s):  
Carboxy Group ◽  
Gel Filtration ◽  
Serine Hydroxymethyltransferase ◽  
Site Directed Mutagenesis ◽  
Mutant Enzyme ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Partial Dissociation ◽  
Tetrameric Form

In an attempt to identify the arginine residue involved in binding of the carboxylate group of serine to mammalian serine hydroxymethyltransferase, a highly conserved Arg-401 was mutated to Ala by site-directed mutagenesis. The mutant enzyme had a characteristic visible absorbance at 425 nm indicative of the presence of bound pyridoxal 5ʹ-phosphate as an internal aldimine with a lysine residue. However, it had only 0.003% of the catalytic activity of the wild-type enzyme. It was also unable to perform reactions with glycine, β-phenylserine or D-alanine, suggesting that the binding of these substrates to the mutant enzyme was affected. This was also evident from the interaction of amino-oxyacetic acid, which was very slow (8.4×10-4 s-1 at 50 μM) for the R401A mutant enzyme compared with the wild-type enzyme (44.6 s-1 at 50 μM). In contrast, methoxyamine (which lacks the carboxy group) reacted with the mutant enzyme (1.72 s-1 at 250 μM) more rapidly than the wild-type enzyme (0.2 s-1 at 250 μM). Further, both wild-type and the mutant enzymes were capable of forming unique quinonoid intermediates absorbing at 440 and 464 nm on interaction with thiosemicarbazide, which also does not have a carboxy group. These results implicate Arg-401 in the binding of the substrate carboxy group. In addition, gel-filtration profiles of the apoenzyme and the reconstituted holoenzyme of R401A and the wild-type enzyme showed that the mutant enzyme remained in a tetrameric form even when the cofactor had been removed. However, the wild-type enzyme underwent partial dissociation to a dimer, suggesting that the oligomeric structure was rendered more stable by the mutation of Arg-401. The increased stability of the mutant enzyme was also reflected in the higher apparent melting temperature (Tm) (61 °C) than that of the wild-type enzyme (56 °C). The addition of serine or serinamide did not change the apparent Tm of R401A mutant enzyme. These results suggest that the mutant enzyme might be in a permanently ‘open’ form and the increased apparent Tm could be due to enhanced subunit interactions.

scholarly journals Expression of Rab3D N135I Inhibits Regulated Secretion of ACTH in AtT-20 Cells

1998 ◽  
Vol 140 (2) ◽  
pp. 305-313 ◽  
Author(s):  
Giulia Baldini ◽  
Giovanna Baldini ◽  
Guangyi Wang ◽  
Mattew Weber ◽  
Marina Zweyer ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Membrane Trafficking ◽  
Hormone Secretion ◽  
Dense Core ◽  
Rab Proteins ◽  
Wild Type ◽  
Small Molecular Weight ◽  
Vesicular Traffic ◽  
Regulated Secretion ◽  
Dense Core Granules

Rab proteins are small molecular weight GTPases that control vesicular traffic in eucaryotic cells. A subset of Rab proteins, the Rab3 proteins are thought to play an important role in regulated exocytosis of vesicles. In transfected AtT-20 cells expressing wild-type Rab3D, we find that a fraction of the protein is associated with dense core granules. In the same cells, expression of a mutated isoform of Rab3D, Rab3D N135I, inhibits positioning of dense core granules near the plasma membrane, blocks regulated secretion of mature ACTH, and impairs association of Rab3A to membranes. Expression of Rab3D N135I does not change the levels of ACTH precursor or the efficiency with which the precursor is processed into ACTH hormone and packaged into dense core granules. We also find that cells expressing mutated Rab3D differentiate to the same extent as untransfected AtT-20 cells. We conclude that expression of Rab3D N135I specifically impairs late membrane trafficking events necessary for ACTH hormone secretion.

p53 domains: structure, oligomerization, and transformation

1994 ◽  
Vol 14 (8) ◽  
pp. 5182-5191
Author(s):  
P Wang ◽  
M Reed ◽  
Y Wang ◽  
G Mayr ◽  
J E Stenger ◽  
...  
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Gel Filtration ◽  
Dominant Negative ◽  
Wild Type ◽  
C Terminus ◽  
Tetramerization Domain ◽  
Wild Type P53 ◽  
Negative Phenotype

Wild-type p53 forms tetramers and multiples of tetramers. Friedman et al. (P. N. Friedman, X. B. Chen, J. Bargonetti, and C. Prives, Proc. Natl. Acad. Sci. USA 90:3319-3323, 1993) have reported that human p53 behaves as a larger molecule during gel filtration than it does during sucrose gradient sedimentation. These differences argue that wild-type p53 has a nonglobular shape. To identify structural and oligomerization domains in p53, we have investigated the physical properties of purified segments of p53. The central, specific DNA-binding domain within murine amino acids 80 to 320 and human amino acids 83 to 323 behaves predominantly as monomers during analysis by sedimentation, gel filtration, and gel electrophoresis. This consistent behavior argues that the central region of p53 is globular in shape. Under appropriate conditions, however, this segment can form transient oligomers without apparent preference for a single oligomeric structure. This region does not enhance transformation by other oncogenes. The biological implications of transient oligomerization by this central segment, therefore, remain to be demonstrated. Like wild-type p53, the C terminus, consisting of murine amino acids 280 to 390 and human amino acids 283 to 393, behaves anomalously during gel filtration and apparently has a nonglobular shape. Within this region, murine amino acids 315 to 350 and human amino acids 323 to 355 are sufficient for assembly of stable tetramers. The finding that murine amino acids 315 to 360 enhance transformation by other oncogenes strongly supports the role of p53 tetramerization in oncogenesis. Amino acids 330 to 390 of murine p53 and amino acids 340 to 393 of human p53, which have been implicated by Sturzbecher et al. in tetramerization (H.-W. Sturzbecher, R. Brain, C. Addison, K. Rudge, M. Remm, M. Grimaldi, E. Keenan, and J. R. Jenkins, Oncogene 7:1513-1523, 1992), do not form stable tetramers under our conditions. Our findings indicate that p53 has at least two autonomous oligomerization domains: a strong tetramerization domain in its C-terminal region and a weaker oligomerization domain in the central DNA binding region of p53. Together, these domains account for the formation of tetramers and multiples of tetramers by wild-type p53. The tetramerization domain is the major determinant of the dominant negative phenotype leading to transformation by mutant p53s.

scholarly journals Polyglycylation of Tubulin Is Essential and Affects Cell Motility and Division in Tetrahymena thermophila

2000 ◽  
Vol 149 (5) ◽  
pp. 1097-1106 ◽  
Author(s):  
Lu Xia ◽  
Bing Hai ◽  
Yan Gao ◽  
Dylan Burnette ◽  
Rupal Thazhath ◽  
...  
Keyword(s):  
Double Mutant ◽  
Tetrahymena Thermophila ◽  
Slow Growth ◽  
Immunochemical Analysis ◽  
Wild Type ◽  
Specific Antibodies ◽  
Essential Function ◽  
Β Tubulin

We analyzed the role of tubulin polyglycylation in Tetrahymena thermophila using in vivo mutagenesis and immunochemical analysis with modification-specific antibodies. Three and five polyglycylation sites were identified at glutamic acids near the COOH termini of α- and β-tubulin, respectively. Mutants lacking all polyglycylation sites on α-tubulin have normal phenotype, whereas similar sites on β-tubulin are essential. A viable mutant with three mutated sites in β-tubulin showed reduced tubulin glycylation, slow growth and motility, and defects in cytokinesis. Cells in which all five polyglycylation sites on β-tubulin were mutated were viable if they were cotransformed with an α-tubulin gene whose COOH terminus was replaced by the wild-type COOH terminus of β-tubulin. In this double mutant, β-tubulin lacked detectable polyglycylation, while the α-β tubulin chimera was hyperglycylated compared with α-tubulin in wild-type cells. Thus, the essential function of polyglycylation of the COOH terminus of β-tubulin can be transferred to α-tubulin, indicating it is the total amount of polyglycylation on both α- and β-tubulin that is essential for survival.

scholarly journals p53 domains: structure, oligomerization, and transformation.

1994 ◽  
Vol 14 (8) ◽  
pp. 5182-5191 ◽  
Author(s):  
P Wang ◽  
M Reed ◽  
Y Wang ◽  
G Mayr ◽  
J E Stenger ◽  
...  
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Gel Filtration ◽  
Dominant Negative ◽  
Wild Type ◽  
C Terminus ◽  
Tetramerization Domain ◽  
Wild Type P53 ◽  
Negative Phenotype

Wild-type p53 forms tetramers and multiples of tetramers. Friedman et al. (P. N. Friedman, X. B. Chen, J. Bargonetti, and C. Prives, Proc. Natl. Acad. Sci. USA 90:3319-3323, 1993) have reported that human p53 behaves as a larger molecule during gel filtration than it does during sucrose gradient sedimentation. These differences argue that wild-type p53 has a nonglobular shape. To identify structural and oligomerization domains in p53, we have investigated the physical properties of purified segments of p53. The central, specific DNA-binding domain within murine amino acids 80 to 320 and human amino acids 83 to 323 behaves predominantly as monomers during analysis by sedimentation, gel filtration, and gel electrophoresis. This consistent behavior argues that the central region of p53 is globular in shape. Under appropriate conditions, however, this segment can form transient oligomers without apparent preference for a single oligomeric structure. This region does not enhance transformation by other oncogenes. The biological implications of transient oligomerization by this central segment, therefore, remain to be demonstrated. Like wild-type p53, the C terminus, consisting of murine amino acids 280 to 390 and human amino acids 283 to 393, behaves anomalously during gel filtration and apparently has a nonglobular shape. Within this region, murine amino acids 315 to 350 and human amino acids 323 to 355 are sufficient for assembly of stable tetramers. The finding that murine amino acids 315 to 360 enhance transformation by other oncogenes strongly supports the role of p53 tetramerization in oncogenesis. Amino acids 330 to 390 of murine p53 and amino acids 340 to 393 of human p53, which have been implicated by Sturzbecher et al. in tetramerization (H.-W. Sturzbecher, R. Brain, C. Addison, K. Rudge, M. Remm, M. Grimaldi, E. Keenan, and J. R. Jenkins, Oncogene 7:1513-1523, 1992), do not form stable tetramers under our conditions. Our findings indicate that p53 has at least two autonomous oligomerization domains: a strong tetramerization domain in its C-terminal region and a weaker oligomerization domain in the central DNA binding region of p53. Together, these domains account for the formation of tetramers and multiples of tetramers by wild-type p53. The tetramerization domain is the major determinant of the dominant negative phenotype leading to transformation by mutant p53s.

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