scholarly journals Chymotryptic digestion of Tetrahymena ciliary dynein. II. Pathway of the degradation of 22S dynein heavy chains.

1987 ◽  
Vol 105 (2) ◽  
pp. 897-901 ◽  
Author(s):  
Y Y Toyoshima
Keyword(s):  
Atpase Activity ◽  
Gel Electrophoresis ◽  
Peptide Mapping ◽  
Preceding Paper ◽  
Degradation Pathway ◽  
Heavy Chains ◽  
Cell Biol ◽  
Dynein Heavy Chains

As shown in the preceding paper (Toyoshima, Y. Y., 1987, J. Cell Biol., 105:887-895) three-headed Tetrahymena 22S dynein consists of three heavy chains (HCs) and is decomposed into two-headed (H) and one-headed (L) fragments by chymotryptic digestion. To accurately determine the presence of multiple ATPases and ultimately the location of various domains, it is necessary to determine the identity of each HC fragment relative to the original HCs in 22S dynein. The degradation pathway of each HC was determined by peptide mapping and immunoblotting. The three HCs (A alpha, A beta, and A gamma) were immunologically different; although SDS-urea gel electrophoresis showed that A gamma HC was apparently resistant to the digestion, actually three distinct HCs contributed to the same band alternately. H fragment was derived from A beta and A gamma HCs, whereas L fragment originated from A alpha HC. Since both fragments were associated with ATPase activity, these results directly demonstrate the presence of multiple ATPase sites in Tetrahymena 22S dynein.

scholarly journals Chymotryptic digestion of Tetrahymena 22S dynein. I. Decomposition of three-headed 22S dynein to one- and two-headed particles.

1987 ◽  
Vol 105 (2) ◽  
pp. 887-895 ◽  
Author(s):  
Y Y Toyoshima
Keyword(s):  
Electron Microscopy ◽  
Gel Electrophoresis ◽  
Gradient Centrifugation ◽  
Sedimentation Coefficient ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Heavy Chains ◽  
Time Courses ◽  
The One ◽  
Dynein Heavy Chains

Molecular composition of Tetrahymena ciliary dynein has been examined by electron microscopy and gel electrophoresis. SDS-urea gel electrophoresis revealed that Tetrahymena 22S dynein contains three (A alpha, A beta, and A gamma) heavy chains whereas 14S dynein contains only one. The molecular masses of 22S and 14S dynein heavy chains were estimated to be approximately 490 and 460 kD, respectively. Electron microscopy of negatively stained specimens showed 22S dynein has three globular heads and thin stalks, whereas 14S dynein consists of a single head. Chymotrypsin digested each of the three 22S dynein heavy chains into large fragments with different time courses. Sucrose density gradient centrifugation separated the digestion products as two peaks. The one with a larger sedimentation coefficient mainly consisted of two-headed particles having binding ability to doublet microtubules, whereas the other with a smaller sedimentation coefficient consisted of only isolated globular particles. Both fractions had ATPase activities. Thus, one active head of 22S dynein can be isolated by chymotrypsin digestion.

scholarly journals The intraflagellar transport dynein complex of trypanosomes is made of a heterodimer of dynein heavy chains and of light and intermediate chains of distinct functions

2014 ◽  
Vol 25 (17) ◽  
pp. 2620-2633 ◽  
Author(s):  
Thierry Blisnick ◽  
Johanna Buisson ◽  
Sabrina Absalon ◽  
Alexandra Marie ◽  
Nadège Cayet ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Intraflagellar Transport ◽  
Retrograde Transport ◽  
High Concentration ◽  
Anterograde Transport ◽  
Heavy Chains ◽  
Cilia And Flagella ◽  
The Stability ◽  
Intermediate Chains ◽  
Dynein Heavy Chains

Cilia and flagella are assembled by intraflagellar transport (IFT) of protein complexes that bring tubulin and other precursors to the incorporation site at their distal tip. Anterograde transport is driven by kinesin, whereas retrograde transport is ensured by a specific dynein. In the protist Trypanosoma brucei, two distinct genes encode fairly different dynein heavy chains (DHCs; ∼40% identity) termed DHC2.1 and DHC2.2, which form a heterodimer and are both essential for retrograde IFT. The stability of each heavy chain relies on the presence of a dynein light intermediate chain (DLI1; also known as XBX-1/D1bLIC). The presence of both heavy chains and of DLI1 at the base of the flagellum depends on the intermediate dynein chain DIC5 (FAP133/WDR34). In the IFT140RNAi mutant, an IFT-A protein essential for retrograde transport, the IFT dynein components are found at high concentration at the flagellar base but fail to penetrate the flagellar compartment. We propose a model by which the IFT dynein particle is assembled in the cytoplasm, reaches the base of the flagellum, and associates with the IFT machinery in a manner dependent on the IFT-A complex.

scholarly journals Quantitation of two endogenous lactose-inhibitable lectins in embryonic and adult chicken tissues.

1982 ◽  
Vol 92 (1) ◽  
pp. 23-27 ◽  
Author(s):  
E C Beyer ◽  
S H Barondes
Keyword(s):  
Gel Electrophoresis ◽  
Peptide Mapping ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cross Reactivity ◽  
Adult Chicken ◽  
Chicken Tissues ◽  
Adult Tissues ◽  
Embryonic Muscle ◽  
The Many ◽  
Kidney Liver

Two lactose-binding lectins from chicken tissues, chicken-lactose-lectin-1 (CLL-1) and chicken-lactose-lectin-11 (CLL-11) were quantified with a radioimmunoassay in extracts of a number of developing and adult chicken tissues. Both lectins could be measured in the same extract without separation, because they showed not significant immunological cross-reactivity. Many embryonic and adult tissues, including brain, heart, intestine, kidney, liver, lung, muscle, pancreas, and spleen, contained one or both lectins, although their concentrations differed markedly. For example, embryonic muscle, the richest source of CLL-1 contained only traces of CLL-11 whereas embryonic kidney, a very rich source of CLL-11 contained substantial CLL-1. In both muscle and kidney, lectin levels in adulthood were much lower than in the embryonic state. In contrast, CLL-1 in liver and CLL-11 in intestine were 10-fold to 30-fold more concentrated in the adult than in the 15-d embryo. CLL-1 and CLL-11 from several tissues were purified by affinity chromatography and their identity in the various tissues was confirmed by polyacrylamide gel electrophoresis, isoelectric focusing, and peptide mapping. The results suggest that these lectins might have different functions in the many developing and adult tissues in which they are found.

scholarly journals The Pathway of Us11-Dependent Degradation of Mhc Class I Heavy Chains Involves a Ubiquitin-Conjugated Intermediate

1999 ◽  
Vol 147 (1) ◽  
pp. 45-58 ◽  
Author(s):  
Caroline E. Shamu ◽  
Craig M. Story ◽  
Tom A. Rapoport ◽  
Hidde L. Ploegh
Keyword(s):  
Mhc Class I ◽  
Heavy Chain ◽  
Control Process ◽  
Degradation Pathway ◽  
Cell System ◽  
Class I ◽  
Permeabilized Cell ◽  
Intact Cells ◽  
Heavy Chains ◽  
Quality Control Process

The human cytomegalovirus protein, US11, initiates the destruction of MHC class I heavy chains by targeting them for dislocation from the ER to the cytosol and subsequent degradation by the proteasome. We report the development of a permeabilized cell system that recapitulates US11-dependent degradation of class I heavy chains. We have used this system, in combination with experiments in intact cells, to identify and order intermediates in the US11-dependent degradation pathway. We find that heavy chains are ubiquitinated before they are degraded. Ubiquitination of the cytosolic tail of heavy chain is not required for its dislocation and degradation, suggesting that ubiquitination occurs after at least part of the heavy chain has been dislocated from the ER. Thus, ubiquitination of the heavy chain does not appear to be the signal to start dislocation. Ubiquitinated heavy chains are associated with membrane fractions, suggesting that ubiquitination occurs while the heavy chain is still bound to the ER membrane. Our results support a model in which US11 co-opts the quality control process by which the cell destroys misfolded ER proteins in order to specifically degrade MHC class I heavy chains.

scholarly journals Tubulin heterogeneity in the trypanosome Crithidia fasciculata.

1984 ◽  
Vol 4 (4) ◽  
pp. 779-790 ◽  
Author(s):  
D G Russell ◽  
D Miller ◽  
K Gull
Keyword(s):  
Gel Electrophoresis ◽  
Peptide Mapping ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
In Vitro Translation ◽  
Two Dimensional ◽  
Post Translational Modification ◽  
Interphase Cell ◽  
Crithidia Fasciculata ◽  
Alpha Tubulin

The interphase cell of Crithidia fasciculata has three discrete tubulin populations: the subpellicular microtubules, the axonemal microtubules, and the nonpolymerized cytoplasmic pool protein. These three tubulin populations were independently and selectively purified, yielding, in each case, microtubule protein capable of self-assembly. All three preparations polymerized to form ribbons and sheets rather than the more usual microtubular structures. Analyses of the tubulin by two-dimensional polyacrylamide gel electrophoresis, isoelectric focusing, and peptide mapping indicated that the beta-tubulin complex remained constant regardless of source but that some heterogeneity was present in the alpha subunit. Cytoplasmic pool alpha tubulins (alpha 1/alpha 2) were the only alpha isotypes in the cytoplasm and also formed most of the alpha tubulin species in the pellicular fraction. Flagellar alpha tubulin (alpha 3) was the sole alpha isotype in the flagella; it appeared in small amounts in the pellicular fraction but was completely absent from the cytoplasm. In vitro translation products from polyadenylated RNA from C. fasciculata were also examined by two-dimensional polyacrylamide gel electrophoresis and possessed a protein corresponding to alpha 1/alpha 2 tubulin but lacked any alpha 3 tubulin. The alpha 3 polypeptide arose from a post-translational modification of a precursor polypeptide not identifiable by two-dimensional polyacrylamide gel electrophoresis as alpha 3. Peptide mapping data indicated that cytoplasmic alpha tubulin is the most likely precursor. These results demonstrate alpha-tubulin heterogeneity in this organism and also how close the relationship between flagellar and cytoskeletal tubulins can be among lower eucaryotes.

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