Chaperonin-mediated folding of vertebrate actin-related protein and gamma-tubulin

Melki, Ronald; Vainberg, IE; Chow, RL; Cowan, NJ

doi:10.1083/jcb.122.6.1301

Cited by 166 publications

(108 citation statements)

References 37 publications

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“…Preparation of unfolded target proteins. Cloned cDNAs encoding the entire coding regions for ,B-actin (11), actinvertebrate actin-related protein (RPV) (26), (x- (47), 3-(48), and y-tubulin (50), TCP-1 (1), cyclin B (36), cap-binding protein (40), c-Myc (37), and p2lras (H-Ras) (25) were expressed as labeled polypeptides in E. coli BL21(DE3) by using pET vectors (43), and the labeled denatured proteins were purified from insoluble inclusion bodies as described before (16,17,31). The radiochemical and biochemical purity of each target protein was determined by analysis on SDS-polyacrylamide gels; this allowed an accurate estimation of specific radioactivity.…”

Section: Methodsmentioning

confidence: 99%

Facilitated folding of actins and tubulins occurs via a nucleotide-dependent interaction between cytoplasmic chaperonin and distinctive folding intermediates.

Melki¹,

Cowan²

1994

Mol. Cell. Biol.

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107

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In the cytoplasm of eukaryotes, the folding of actins and tubulins is facilitated via interaction with a heteromeric toroidal complex (cytoplasmic chaperonin). The folding reaction consists of the formation of a binary complex between the unfolded target protein and the chaperonin, followed by the ultimate release of the native polypeptide in an ATP-dependent reaction. Here we show that the mitochondrial chaperonin (cpn6O) and the cytoplasmic chaperonin both recognize a range of target proteins with different relative affinities; however, the cytoplasmic chaperonin shows the highest affinity for intermediates derived from unfolded tubulins and actins. These high-affinity actin and tubulin folding intermediates are distinct from the "molten globule" intermediates formed by noncytoskeletal target proteins in that they form relatively slowly. We show that the interaction between cytoplasmic chaperonin and unfolded target proteins depends on the chaperonin being in its ADP-bound state and that the release of the target protein occurs after a transition of the chaperonin to the ATP-bound state. Our data suggest a model in which ATP hydrolysis acts as a switch between conformational forms of the cytoplasmic chaperonin that interact either strongly or weakly with unfolded substrates.The biological function of most, if not all, proteins depends critically upon their three-dimensional structure. Although the information contained in the linear sequence of amino acids is thought to be sufficient. to specify this structure (2, 24), the correct folding of many proteins is not a spontaneous process under physiological conditions; rather, there is a requirement for interaction with a class of proteins or protein complexes known as molecular chaperones (reviewed in references 14, 18, and 39). In particular, one class of chaperones (typified by GroEL in prokaryotes) consist of multisubunit toroidal ring assemblies and are believed to function by providing a sequestered environment in which aberrant folding and aggregation are prevented; the correctly folded polypeptide is ultimately discharged in an Mg-ATP-dependent reaction. These toroidal structures are termed chaperonins (6,10,19,28,34,35,46). Actin and tubulin are the major soluble cytoplasmic proteins in eukaryotic cells and are the subunits from which actin filaments and microtubules are assembled. The folding of actins, tubulins, and actin-and tubulin-related proteins is facilitated by a chaperonin (15-17, 31, 49) that differs from its prokaryotic and mitochondrial homologs in that it is heteromeric: the multisubunit toroidal structure is assembled from eight different (though related) polypeptides (15, 38), one of which is the t-complex polypeptide TCP-1 (27, 42). As is the case for other chaperonins, the cytoplasmic chaperonin forms a stable binary complex with unfolded target polypeptides in the absence of Mg-ATP, and the hydrolysis of Mg-ATP is absolutely required for the generation of folded proteins (15-17). However, an important difference exists between the mecha...

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Section: Methodsmentioning

confidence: 99%

Facilitated folding of actins and tubulins occurs via a nucleotide-dependent interaction between cytoplasmic chaperonin and distinctive folding intermediates.

Melki¹,

Cowan²

1994

Mol. Cell. Biol.

Self Cite

107

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“…UXT and co-factor D (Melki et al, 1993;Zhao et al, 2005;Cunningham and Kahn, 2008). Interestingly, co-factor D contains a series of HEAT sequence repeats, reminiscent of the HAT repeats found in HCA66, and silencing also results in spindle pole defects (Cunningham and Kahn, 2008).…”

Section: Journal Of Cell Science 122 (8)mentioning

confidence: 99%

Stability of the small γ-tubulin complex requires HCA66, a protein of the centrosome and the nucleolus

Fant

Gnadt

Haren

et al. 2009

Journal of Cell Science

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“…Another striking difference between the two chaperonins is their different substrate specificity. In contrast to GroEL, which recognizes and assists folding of a broad range of non-native protein substrates, CCT has been demonstrated to facilitate folding of relatively few proteins: actin, tubulin, actin-and tubulin-related proteins, and luciferase (Gao et al, 1992;Frydman et al, 1992;Yaffe et al, 1992;Melki et al, 1993). It has been suggested that the subunits of the heterooligomeric cytosolic chaperonin may have evolved to achieve high specificity for cytoskeletal proteins.…”

mentioning

confidence: 99%

MgATP binding to the nucleotide‐binding domains of the eukaryotic cytoplasmic chaperonin induces conformational changes in the putative substrate‐binding domains

et al. 1998

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The eukaryotic cytosolic chaperonins are large heterooligomeric complexes with a cylindrical shape, resembling that of the homooligomeric bacterial counterpart, GroEL. In analogy to GroEL, changes in shape of the cytosolic chaperonin have been detected in the presence of MgATP using electron microscopy but, in contrast to the nucleotide-induced conformational changes in GroEL, no details are available about the specific nature of these changes. The present study identifies the structural regions of the cytosolic chaperonin that undergo conformational changes when MgATP binds to the nucleotide binding domains. It is shown that limited proteolysis with trypsin in the absence of MgATP cleaves each of the eight subunits approximately in half, generating two fragments of -30 kDa. Using mass spectrometry (MS) and N-terminal sequence analysis, the cleavage is found to occur in a narrow span of the amino acid sequence, corresponding to the peptide binding regions of GroEL and to the helical protrusion, recently identified in the structure of the substrate binding domain of the archeal group I1 chaperonin. This proteolytic cleavage is prevented by MgATP but not by ATP in the absence of magnesium, ATP analogs (MgATPyS and MgAMP-PNP) or MgADP. These results suggest that, in analogy to GroEL, binding of MgATP to the nucleotide binding domains of the cytosolic chaperonin induces long range conformational changes in the polypeptide binding domains. It is postulated that despite their different subunit composition and substrate specificity, group I and group I1 chaperonins may share similar, functionally-important, conformational changes. Additional conformational changes are likely to involve a flexible helix-loop-helix motif, which is characteristic for all group I1 chaperonins. Keywords: CCT; limited proteolysis; long-range conformational changes; mass spectrometryChaperonins are a family of large, sequence-related, oligomeric complexes found in chloroplasts, mitochondria and eubacteria (Group I chaperonins, or GroE subclass, represented by GroEL), and in archaebacteria and eukaryotic cytosol (Group I1 chaperonins or TCP-I subclass) (Ellis, 1996). The structure of the chaperonins consists of two stacked rings of 7-9 subunits, with a large

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Chaperonin-mediated folding of vertebrate actin-related protein and gamma-tubulin

Cited by 166 publications

References 37 publications

Facilitated folding of actins and tubulins occurs via a nucleotide-dependent interaction between cytoplasmic chaperonin and distinctive folding intermediates.

Facilitated folding of actins and tubulins occurs via a nucleotide-dependent interaction between cytoplasmic chaperonin and distinctive folding intermediates.

Stability of the small γ-tubulin complex requires HCA66, a protein of the centrosome and the nucleolus

MgATP binding to the nucleotide‐binding domains of the eukaryotic cytoplasmic chaperonin induces conformational changes in the putative substrate‐binding domains

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