Newly-synthesized beta-tubulin demonstrates domain-specific interactions with the cytosolic chaperonin.
Tubulin folding requires two chaperone systems, i.e., the 900 kDa cytosolic chaperonin referred to as the TCP-1 complex or TRiC which facilitates folding of the alpha- and beta-tubulin subunits and a ca. 180 kDa complex which facilitates further assembly into heterodimer. beta-Tubulin mutants were expressed in rabbit reticulocyte lysates, and the effect of C-terminal, N-terminal, and internal deletions on the binding of beta-tubulin polypeptides to the 900 and 180 kDa complexes was ascertained. Proteolytic studies of chaperonin-bound beta-tubulin were also implemented. These studies support the concept of quasi-native chaperonin-bound intermediates [Tian et al. J. Biol. Chem. (1995) 270, 1-4]. Three "domains" similar in size to the domains in the native protein were implicated in facilitated folding: i.e., an internal or "M-domain" composed of residues approximately 140-260 which binds to TRiC; a "C-domain" composed of residues approximately 300-445 which interacts less strongly with TRiC and may contain regulatory sequences for tubulin release from the chaperonin; and an "N-domain" composed of residues approximately 1-140 which apparently does not interact with TRiC but does interact with the 180 kDa complex. The major TRiC-interacting region, residues approximately 150-350 (the "interactive core"), overlapped portions of the M- and C-domains and included a putative hydrophobic-rich interdomain segment which may be a preferential site of interaction with TRiC. This segment may also be important for microtubule assembly and/or tubulin dimer formation. Removal of two residues from the N-terminal end or ca. 27 residues from the C-terminal and caused the polypeptide to arrest on TRiC. It is proposed that N- and C-terminal regions of beta-tubulin structurally interact with TRiC-binding region approximately 150-350 to inhibit binding to TRiC.
Dobrzynski
J K
JK
Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Sternlicht
M L
ML
Farr
G W
GW
Sternlicht
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D056404:Chaperonin Containing TCP-1; D018833:Chaperonins / Q000378:metabolism*; D002645:Chickens; D002850:Chromatography, Gel; D002918:Chymotrypsin / Q000378:metabolism; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D047908:Intracellular Signaling Peptides and Proteins; D008869:Microtubule-Associated Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009154:Mutation / Q000235:genetics; D009687:Nuclear Proteins / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D011233:Precipitin Tests; D014176:Protein Biosynthesis / Q000235:genetics; D017510:Protein Folding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion / Q000235:genetics; D014404:Tubulin / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases; D056427:t-Complex Genome Region
D047908:Intracellular Signaling Peptides and Proteins; D008869:Microtubule-Associated Proteins; D009687:Nuclear Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014404:Tubulin; C116196:PPP1R11 protein, human; D044767:Ubiquitin-Protein Ligases; D002918:Chymotrypsin; D056404:Chaperonin Containing TCP-1; D018833:Chaperonins