Tubulin in sea urchin embryonic cilia: post-translational modifications during regeneration

1992 ◽  
Vol 101 (4) ◽  
pp. 837-845 ◽  
Author(s):  
R.E. Stephens
Keyword(s):  
Monoclonal Antibody ◽  
Sea Urchin ◽  
Growth Phase ◽  
Linear Growth ◽  
Major Protein ◽  
Post Translational Modifications ◽  
Alpha Tubulin ◽  
Ciliary Membrane ◽  
Membrane Matrix ◽  
Matrix Fraction

Tubulin is the major protein found in the membrane/periaxonemal matrix fraction of mature sea urchin embryonic cilia but its distribution and possible function during ciliary assembly are unknown. Hypertonic salt may be used to deciliate the embryos, allowing synchronous regrowth of cilia and subsequent deciliation of the regenerating embryos at various times. During the earliest stages of regeneration, the amounts of tubulin in the axoneme and membrane/matrix fractions are nearly equal, but the proportion of tubulin in the axoneme fraction increases coincident with the quasi-linear growth phase while the membrane/matrix tubulin remains constant. Antibodies to tyrosinated and detyrosinated alpha-tubulin show that both the membrane/matrix and axonemal tubulin fractions are primarily unmodified (i.e. tyrosinated) at the earliest stages of regeneration but are progressively and equally detyrosinated coincident with regeneration, approaching a final level of 50% C-terminal Glu. A monoclonal antibody to acetylated alpha-tubulin reveals that both tubulin fractions are equally and maximally acetylated at relatively early stages of regeneration. In contrast, three-times-repolymerized tubulin from either unfertilized eggs or midgastrula embryos is primarily tyrosinated (greater than 97%) and not detectably acetylated. These data suggest that membrane/matrix tubulin is a precursor to axonemal tubulin and that acetylation and detyrosination may be involved in partitioning tubulin among cytoplasmic, ciliary membrane/matrix, and 9 + 2 compartments.

Tubulin and tektin in sea urchin embryonic cilia: pathways of protein incorporation during turnover and regeneration

1994 ◽  
Vol 107 (2) ◽  
pp. 683-692 ◽  
Author(s):  
R.E. Stephens
Keyword(s):  
Sea Urchin ◽  
Relative Concentration ◽  
Major Protein ◽  
Membrane Matrix ◽  
Low Concentrations ◽  
Architectural Proteins ◽  
Uniform Labeling ◽  
Matrix Fraction ◽  
Protein Incorporation ◽  
Microtubule Doublet

Axonemal precursor tubulin is the major protein component of the detergent-soluble membrane/matrix fraction of sea urchin embryonic cilia. Its unusual abundance may reflect the rapid turnover of these cilia, a process that is further documented here. However, whether during induced regeneration or normal turnover and growth, most other newly synthesized axonemal proteins are not detectable in the membrane/matrix fraction, raising the question of how non-tubulin precursors transit the growing cilium to the distal tip where assembly is generally thought to occur. Three potential explanations were considered: (1) the assembly of these components is proximal; (2) their relative concentration is too low to detect; or (3) tubulin alone is conveyed via a membrane/matrix pathway while most other axonemal proteins are transported in association with the axoneme. Light microscope autoradiography of axonemes pulse-chase labeled with [3H]leucine showed relatively uniform labeling, with no evidence for proximal incorporation. Fully grown cilia and cilia at early stages of regeneration were isolated from labeled embryos, fractionated into membrane/matrix, axonemal tubulin and architectural remnant components, and their labeled protein compositions were compared. Heavily labeled axonemal proteins, most notably the integral microtubule doublet component tektin-A, were not detected in the membrane/matrix fraction of emerging cilia, even though nearly half of the total ciliary tubulin appeared in that fraction, arguing against membrane-associated or soluble matrix transit for the architectural proteins at low concentrations. However, after thermal fractionation of axonemes from growing cilia, labeled proteins characteristic of the architectural remnant dominated the solubilized microtubule fraction, supporting axoneme-associated transport of the non-tubulin proteins during growth, in contrast to a membrane/matrix pathway for tubulin.

Tubulin in sea urchin embryonic cilia: characterization of the membrane-periaxonemal matrix

1991 ◽  
Vol 100 (3) ◽  
pp. 521-531 ◽  
Author(s):  
R.E. Stephens
Keyword(s):  
Membrane Protein ◽  
Sea Urchin ◽  
Posttranslational Modifications ◽  
Calcium Binding ◽  
Specific Activity ◽  
Cross Reactivity ◽  
Alpha Tubulin ◽  
Thaw Cycle ◽  
Freeze Thaw Cycle ◽  
Matrix Fraction

When the membranes of isolated embryonic cilia from three species of sea urchin are detergent-solubilized, the major proteins found are two equi-molar polypeptides comigrating with tubulin subunits. Cross-reactivity with a variety of tubulin antibodies confirms their identity. Calmodulin and other calcium-binding proteins are prominent minor constituents of the extract. Removal of the solubilizing detergent by adsorption to polystyrene beads, followed by a freeze-thaw cycle, produces membrane leaflets and vesicles of uniform bouyant density. Such reconstituted membranes incorporate most of the tubulin and minor proteins but not calmodulin. Equivalent cross-reactivity with antibodies to acetylated or detyrosinated alpha-tubulin indicates that the tubulin derived from the membrane-periaxonemal matrix and axoneme are indistinguishable from each other in terms of these posttranslational modifications but are distinct from the mainly unmodified tubulin of the embryonic cytoplasm. Pulse labeling with [3H]palmitate does not label either tubulin subunit but acylation does occur on a 190 × 10(3) Mr membrane protein. Its specific activity is essentially the same whether label is applied to embryos with existing or regenerating cilia, suggesting rapid ciliary membrane protein exchange or physical turnover. Using pulse-chase labeling with [3H]leucine during steady-state ciliary growth or induced regeneration in both normal and zinc-animalized embryos, the specific activity of the membrane-periaxonemal matrix-derived tubulin is initially higher than that of the axoneme but the degree of labeling equalizes in successive regenerations, consistent with derivation from a common pool. Many heavily labeled axonemal architectural proteins, such as tektin-A, are not reflected in the membrane-periaxonemal matrix fraction, suggesting that this fraction is not simply a pool of unassembled axonemal precursors.

Reading the phosphorylation code: binding of the 14-3-3 protein to multivalent client phosphoproteins

2020 ◽  
Vol 477 (7) ◽  
pp. 1219-1225 ◽  
Author(s):  
Nikolai N. Sluchanko
Keyword(s):  
Protein Function ◽  
Intrinsically Disordered Protein ◽  
Structural Basis ◽  
Major Protein ◽  
Post Translational Modifications ◽  
Protein Protein Interaction ◽  
Intrinsically Disordered ◽  
Biochemical Journal ◽  
Multiple Binding ◽  
And Control

Many major protein–protein interaction networks are maintained by ‘hub’ proteins with multiple binding partners, where interactions are often facilitated by intrinsically disordered protein regions that undergo post-translational modifications, such as phosphorylation. Phosphorylation can directly affect protein function and control recognition by proteins that ‘read’ the phosphorylation code, re-wiring the interactome. The eukaryotic 14-3-3 proteins recognizing multiple phosphoproteins nicely exemplify these concepts. Although recent studies established the biochemical and structural basis for the interaction of the 14-3-3 dimers with several phosphorylated clients, understanding their assembly with partners phosphorylated at multiple sites represents a challenge. Suboptimal sequence context around the phosphorylated residue may reduce binding affinity, resulting in quantitative differences for distinct phosphorylation sites, making hierarchy and priority in their binding rather uncertain. Recently, Stevers et al. [Biochemical Journal (2017) 474: 1273–1287] undertook a remarkable attempt to untangle the mechanism of 14-3-3 dimer binding to leucine-rich repeat kinase 2 (LRRK2) that contains multiple candidate 14-3-3-binding sites and is mutated in Parkinson's disease. By using the protein-peptide binding approach, the authors systematically analyzed affinities for a set of LRRK2 phosphopeptides, alone or in combination, to a 14-3-3 protein and determined crystal structures for 14-3-3 complexes with selected phosphopeptides. This study addresses a long-standing question in the 14-3-3 biology, unearthing a range of important details that are relevant for understanding binding mechanisms of other polyvalent proteins.

Microtubules rich in modified alpha-tubulin characterize the tail processes of motile fibroblasts

1989 ◽  
Vol 94 (2) ◽  
pp. 227-236
Author(s):  
A.R. Prescott ◽  
M. Vestberg ◽  
R.M. Warn
Keyword(s):  
Cell Motility ◽  
Immunofluorescence Microscopy ◽  
Fibroblast Cell ◽  
Microscopy Study ◽  
Post Translational Modifications ◽  
Alpha Tubulin ◽  
Major Species ◽  
Cytoplasmic Tails ◽  
Nih 3T3

The organisation of microtubules rich in post-translationally modified alpha-tubulin has been investigated in a fibroblast cell line (NIH-3T3-T15) that can be reversibly transformed. An immunofluorescence microscopy study of the static non-transformed cells has revealed a central distribution of wavy microtubules showing post-translational modifications. When transformed there is a marked increase in cell motility and the appearance of long thin cytoplasmic ‘tails’. These tails have been found to contain conspicuous bundles of post-translationally modified microtubules that run down the length of the processes and terminate close to the plasmalemma. Both detyrosinated and acetylated alpha-tubulin are present as major species in these modified microtubules. Such a pattern of modified microtubules is only occasionally seen in the untransformed NIH-3T3-T15 cells. We have also found them to be present in other transformed fibroblast lines. The presence of bundles of microtubules rich in modified alpha-tubulin in the cell tails is correlated with a marked reduction in the numbers of F-actin stress fibres. The possible role of these modified stable microtubules in cell motility is discussed.

Centrosomal components immunologically related to tektins from ciliary and flagellar microtubules

1994 ◽  
Vol 107 (8) ◽  
pp. 2095-2105 ◽  
Author(s):  
W. Steffen ◽  
E.A. Fajer ◽  
R.W. Linck
Keyword(s):  
Cell Cycle ◽  
Sea Urchin ◽  
Cho Cells ◽  
Mammalian Cells ◽  
Polyclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Immunoblot Analysis ◽  
Surf Clam ◽  
Alpha Tubulin ◽  
Sea Urchin Sperm

Centrosomes are critical for the nucleation and organization of the microtubule cytoskeleton during both interphase and cell division. Using antibodies raised against sea urchin sperm flagellar microtubule proteins, we characterize here the presence and behavior of certain components associated with centrosomes of the surf clam Spisula solidissima and cultured mammalian cells. A Sarkosyl detergent-resistant fraction of axonemal microtubules was isolated from sea urchin sperm flagella and used to produce monoclonal antibodies, 16 of which were specific- or cross-specific for the major polypeptides associated with this microtubule fraction: tektins A, B and C, acetylated alpha-tubulin, and 77 and 83 kDa polypeptides. By 2-D isoelectric focussing/SDS polyacrylamide gel electrophoresis the tektins separate into several polypeptide spots. Identical spots were recognized by monoclonal and polyclonal antibodies against a given tektin, indicating that the different polypeptide spots are isoforms or modified versions of the same protein. Four independently derived monoclonal anti-tektins were found to stain centrosomes of S. solidissima oocytes and CHO and HeLa cells, by immunofluorescence microscopy. In particular, the centrosome staining of one monoclonal antibody specific for tektin B (tekB3) was cell-cycle-dependent for CHO cells, i.e. staining was observed only from early prometaphase until late anaphase. By immuno-electron microscopy tekB3 specifically labeled material surrounding the centrosome, whereas a polyclonal anti-tektin B recognized centrioles as well as the centrosomal material throughout the cell cycle. Finally, by immunoblot analysis tekB3 stained polypeptides of 48–50 kDa in isolated spindles and centrosomes from CHO cells.

scholarly journals Post-Translational Modifications of Extracellular Proteasome

Molecules ◽  
2020 ◽  
Vol 25 (15) ◽  
pp. 3504 ◽  
Author(s):  
Anna S. Tsimokha ◽  
Tatiana O. Artamonova ◽  
Egor E. Diakonov ◽  
Mikhail A. Khodorkovskii ◽  
Alexey N. Tomilin
Keyword(s):  
Extracellular Vesicles ◽  
Extracellular Space ◽  
Neurological Diseases ◽  
Prognostic Significance ◽  
Ubiquitin Proteasome System ◽  
Major Protein ◽  
Ion Cyclotron Resonance ◽  
Post Translational Modifications ◽  
Essential Components ◽  
Proteasome Subunits

The ubiquitin-proteasome system (UPS) is one of the major protein degradation pathways in eukaryotic cells. Abnormal functioning of this system has been observed in cancer and neurological diseases. The 20S proteasomes, essential components of the UPS, are present not only within the cells but also in the extracellular space, and their concentration in blood plasma has been found to be elevated and dependent upon the disease state, being of prognostic significance in patients suffering from cancer, liver diseases, and autoimmune diseases. However, functions of extracellular proteasomes and mechanisms of their release by cells remain largely unknown. The main mechanism of proteasome activity regulation is provided by modulation of their composition and post-translational modifications (PTMs). Moreover, diverse PTMs of proteins are known to participate in the loading of specific elements into extracellular vesicles. Since previous studies have revealed that the transport of extracellular proteasomes may occur via extracellular vesicles, we have set out to explore the PTMs of extracellular proteasomes in comparison to cellular counterparts. In this work, cellular and extracellular proteasomes were affinity purified and separated by SDS-PAGE for subsequent trypsinization and matrix-assisted laser desorption/ionization (MALDI) Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) analysis. In total, we could identify 64 and 55 PTM sites in extracellular and cellular proteasomes, respectively, including phosphorylation, ubiquitination, acetylation, and succinylation. We observed novel sites of acetylation at K238 and K192 of the proteasome subunits β2 and β3, respectively, that are specific for extracellular proteasomes. Moreover, cellular proteasomes show specific acetylation at K227 of α2 and ubiquitination at K201 of β3. Interestingly, succinylation of β6 at the residue K228 seems not to be present exclusively in extracellular proteasomes, whereas both extracellular and cellular proteasomes may also be acetylated at this site. The same situation takes place at K201 of the β3 subunit where ubiquitination is seemingly specific for cellular proteasomes. Moreover, crosstalk between acetylation, ubiquitination, and succinylation has been observed in the subunit α3 of both proteasome populations. These data will serve as a basis for further studies, aimed at dissection of the roles of extracellular proteasome-specific PTMs in terms of the function of these proteasomes and mechanism of their transport into extracellular space.

A monoclonal antibody inhibits calcium accumulation and skeleton formation in cultured embryonic cells of the sea urchin

Cell ◽  
1985 ◽  
Vol 41 (2) ◽  
pp. 639-648 ◽  
Author(s):  
D CARSON ◽  
M FARACHACH ◽  
D EARLES ◽  
G DECKER ◽  
W LENNARZ
Keyword(s):  
Monoclonal Antibody ◽  
Sea Urchin ◽  
Embryonic Cells ◽  
Calcium Accumulation ◽  
Skeleton Formation

scholarly journals BIOCHEMICAL AND ULTRASTRUCTURAL PROPERTIES OF A MITOCHONDRIAL INNER MEMBRANE FRACTION DEFICIENT IN OUTER MEMBRANE AND MATRIX ACTIVITIES

1970 ◽  
Vol 45 (2) ◽  
pp. 291-305 ◽  
Author(s):  
T. L. Chan ◽  
John W. Greenawalt ◽  
Peter L. Pedersen
Keyword(s):  
Outer Membrane ◽  
Membrane Fraction ◽  
Phosphorylation Site ◽  
Membrane Vesicles ◽  
Rat Liver Mitochondria ◽  
Mitochondrial Activity ◽  
Inner Membrane ◽  
Membrane Matrix ◽  
Matrix Fraction ◽  
Stimulation Of

Treatment of the inner membrane matrix fraction of rat liver mitochondria with the nonionic detergent Lubrol WX solubilized about 70% of the total protein and 90% or more of the following matrix activities: malate dehydrogenase, glutamate dehydrogenase, and isocitrate dehydrogenase (NADP). The Lubrol-insoluble fraction was enriched in cytochromes, phospholipids, and a Mg++-stimulated ATPase activity. Less than 2% of the total mitochondrial activity of monoamine oxidase, an outer membrane marker, or adenylate kinase, an intracristal space marker could be detected in this inner membrane fraction. Electron micrographs of negatively stained preparations showed vesicles (≤0.4 µ diameter) literally saturated on the periphery with the 90 A ATPase particles. These inner membrane vesicles, which appeared for the most part to be inverted with respect to the normal inner membrane configuration in intact mitochondria, retained the succinicoxidase portion of the electron-transport chain, an intact phosphorylation site II with a high affinity for ADP, and the capacity to accumulate Ca++. A number of biochemical properties characteristic of intact mitochondria and the inner membrane matrix fraction, however, were either absent or markedly deficient in the inner membrane vesicles. These included stimulation of respiration by either ADP or 2,4-dinitrophenol, oligomycin-sensitive ADP-ATP exchange activity, atractyloside sensitivity of adenine nucleotide requiring reactions, and a stimulation of the Mg++-ATPase by 2,4-dinitrophenol.

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