Arthrin, a myofibrillar protein of insect flight muscle, is an actin-ubiquitin conjugate

Ball, Elizabeth; Karlik, C C; Beall, Clifford J.; Saville, Donna; Sparrow, John C.; Bullard, Belinda; Fyrberg, Eric

doi:10.1016/0092-8674(87)90149-8

Cited by 208 publications

(101 citation statements)

References 32 publications

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“…In the absence of Ca 2ϩ , some monoubiquitination of CaM occurred (Fig. 3c), although most data suggest that the attachment of one Ub to a substrate does not generally promote its degradation (31,49). The monoubiquitination of CaM probably involves Lys-115, a residue only available for ubiquitination in bacterially expressed CaMs (as used here) and in Dictyostelium discoideum (50).…”

Section: Discussionmentioning

confidence: 99%

Ca2+-free Calmodulin and Calmodulin Damaged byin Vitro Aging Are Selectively Degraded by 26 S Proteasomes without Ubiquitination

Tarcsa¹,

Szymańska²,

Lecker³

et al. 2000

Journal of Biological Chemistry

104

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The ubiquitin-proteasome pathway is believed to selectively degrade post-synthetically damaged proteins in eukaryotic cells. To study this process we used calmodulin (CaM) as a substrate because of its importance in cell regulation and because it acquires isoaspartyl residues in its Ca 2؉ -binding regions both in vivo and after in vitro "aging" (incubation for 2 weeks without Ca 2؉ ). When microinjected into Xenopus oocytes, in vitro aged CaM was degraded much faster than native CaM by a proteasome-dependent process. Similarly, in HeLa cell extracts aged CaM was degraded at a higher rate, even though it was not conjugated to ubiquitin more rapidly than the native species. Ca 2؉ stimulated the ubiquitination of both species, but inhibited their degradation. Thus, for CaM, ubiquitination and proteolysis appear to be dissociated. Accordingly, purified muscle 26 S proteasomes could degrade aged CaM and native Ca 2؉ -free (apo) CaM without ubiquitination. Addition of Ca 2؉ dramatically reduced degradation of the native molecules but only slightly reduced the breakdown of the aged species. Thus, upon Ca 2؉ binding, native CaM assumes a non-degradable conformation, which most of the agedamaged species cannot assume. Thus, flexible conformations, as may arise from age-induced damage or the absence of ligands, can promote degradation directly by the proteasome without ubiquitination.

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Section: Discussionmentioning

confidence: 99%

Ca2+-free Calmodulin and Calmodulin Damaged byin Vitro Aging Are Selectively Degraded by 26 S Proteasomes without Ubiquitination

Tarcsa¹,

Szymańska²,

Lecker³

et al. 2000

Journal of Biological Chemistry

104

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“…The mAb recognizing the 6xHis tag detected not only the prominent band representing tagged Act88F, but an additional tagged protein, which migrates with an apparent molecular mass of ϳ55 kDa ( Figure 2B, left). Most likely, this band corresponds to 6xHis tagged arthrin, the ubiquitinated form of Act88F, which is typically present in insect IFM at the ratio of one arthrin molecule to six actin molecules (Ball et al, 1987). The P5D4 mAb detected three bands with closely related molecular weights corresponding to actin and two bands representing tagged arthrin ( Figure 2B, right).…”

Section: Expression Of Tagged Act88f Protein In Ifmmentioning

confidence: 99%

“…However, in these experiments the large excess of unmodified endogenous actin is likely to overpower the properties of the modified recombinant actin. To rule out any dominant effects of unmodified endogenous over introduced actin, we have taken advantage of the indirect flight muscle (IFM) of Drosophila melanogaster, which allowed us to unambiguously analyze the consequences of epitope tagging the IFM-specific Act88F actin on muscle structure and function in its bona fide environment.Of the six actin genes in Drosophila, Act88F is expressed only in the IFM, where it is the sole actin isoform found (Fyrberg et al, 1983;Ball et al, 1987). Null mutations of the Act88F gene have yielded strains, for example, KM88 , which because of the lack of endogenous Act88F actin in the IFM are flightless but otherwise perfectly viable.…”

mentioning

confidence: 99%

Differential Epitope Tagging of Actin in TransformedDrosophilaProduces Distinct Effects on Myofibril Assembly and Function of the Indirect Flight Muscle

Brault¹,

Sauder

Reedy

et al. 1999

MBoC

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We have tested the impact of tags on the structure and function of indirect flight muscle (IFM)-specific Act88F actin by transforming mutant Drosophila melanogaster, which do not express endogenous actin in their IFMs, with tagged Act88F constructs. Epitope tagging is often the method of choice to monitor the fate of a protein when a specific antibody is not available. Studies addressing the functional significance of the closely related actin isoforms rely almost exclusively on tagged exogenous actin, because only few antibodies exist that can discriminate between isoforms. Thereby it is widely presumed that the tag does not significantly interfere with protein function. However, in most studies the tagged actin is expressed in a background of endogenous actin and, as a rule, represents only a minor fraction of the total actin. The Act88F gene encodes the only Drosophila actin isoform exclusively expressed in the highly ordered IFM. Null mutations in this gene do not affect viability, but phenotypic effects in transformants can be directly attributed to the transgene. Transgenic flies that express Act88F with either a 6x histidine tag or an 11-residue peptide derived from vesicular stomatitis virus G protein at the C terminus were flightless. Overall, the ultrastructure of the IFM resembled that of the Act88F null mutant, and only low amounts of C-terminally tagged actins were found. In contrast, expression of N-terminally tagged Act88F at amounts comparable with that of wild-type flies yielded fairly normal-looking myofibrils and partially reconstituted flight ability in the transformants. Our findings suggest that the N terminus of actin is less sensitive to modifications than the C terminus, because it can be tagged and still polymerize into functional thin filaments. INTRODUCTIONActins, a highly conserved family of cytoplasmic proteins, are among the most abundant proteins in eukaryotic cells. As a major component of the cytoskeleton, they control shape and motility in nonmuscle cells. In muscle, actin assembles into thin filaments, which together with interdigitating myosin thick filaments provide the framework for muscle contraction.Many organisms synthesize multiple isoforms of actin that are very similar in amino acid sequence even within the same cell. The differential expression of distinct actins as well as the high conservation of specific isoforms across species emphasize the functional importance of isoforms. In the case of actin, the question of how structure determines function appears to be particularly challenging. Considerable efforts have been made to understand how the different isoforms fulfill their various functions despite their extremely high sequence identity, and yet the basis of their functional diversity remains elusive. § Corresponding author. E-mail address: Schoenenberg@ ubaclu.unibas.ch. © 1999 by The American Society for Cell Biology 135Studying the specific role of a particular actin isoform has always been hampered by the difficulty of discriminating between the introduced a...

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“…variety of in vivo substrates of the ubiquitin conjugating system have been identified, including histones [10], actin [11], cytlins [12], the tumor suppressor p53 [13,14], MATch2 transcr ption repressor [15], cell surface receptors [16][17][18], c-jun [19] and NF~cB [20]. Some ubiquitin conjugating enzymes are able to transfer ubiquitin to the model substrate histone in vitro without any accessory factors, such as E3s.…”

Section: Introductionmentioning

confidence: 99%

Characterization of functionally independent domains in the human ubiquitin conjugating enzyme UbcH2

et al. 1995

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UbcH2 encodes a human ubiquitin conjugating enzyme (E2) able to conjugate ubiquitin to histone H2A in an E3 independent manner in vitro, which indicates that UbcH2 directly interacts with its substrates. To identify parts of the enzyme that are capable of binding H2A, we expressed several deletion mutants of UbcH2 in E. coli and tested the ability of the affinity purified mutant proteins to ubiquitinate H2A in the presence of bacterial expressed E1 and ubiquitin. With this in vitro assay we identified a C-terminal part of UbcH2 to be important for the interaction with H2A. Transfer of this C-terminal domain to another human E2, which is unable to catalyze ubiquitination of histories, leads to a fully active hybrid human ubiquitin conjugating enzyme capable of H2A ubiquitination. These results demonstrate that UbcH2 consists of two functionally independent domains. A N-terminal core domain with ubiquitin conjugating activity, and a C-terminal domain which interacts with substrate proteins.

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Arthrin, a myofibrillar protein of insect flight muscle, is an actin-ubiquitin conjugate

Cited by 208 publications

References 32 publications

Ca2+-free Calmodulin and Calmodulin Damaged byin Vitro Aging Are Selectively Degraded by 26 S Proteasomes without Ubiquitination

Ca2+-free Calmodulin and Calmodulin Damaged byin Vitro Aging Are Selectively Degraded by 26 S Proteasomes without Ubiquitination

Differential Epitope Tagging of Actin in TransformedDrosophilaProduces Distinct Effects on Myofibril Assembly and Function of the Indirect Flight Muscle

Characterization of functionally independent domains in the human ubiquitin conjugating enzyme UbcH2

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