Selected Subunits of the Cytosolic Chaperonin Associate with Microtubules Assembled in Vitro

Abstract

The molecular chaperone activities of the only known chaperonin in the eukaryotic cytosol (cytosolic chaperonin containing T-complex polypeptide 1 (CCT)) appear to be relatively specialized; the main folding substrates in vivo and in vitro are identified as tubulins and actins. CCT is unique among chaperonins in the complexity of its hetero-oligomeric structure, containing eight different, although related, gene products. In addition to their known ability to bind to and promote correct folding of newly synthesized and denatured tubulins, we show here that CCT subunits ␣, ␥, , and also associated with in vitro assembled microtubules, i.e. behaved as microtubule-associated proteins. This nucleotide-dependent association between microtubules and CCT polypeptides (K d ϳ 0.1 M CCT subunit) did not appear to involve whole oligomeric chaperonin particles, but rather free CCT subunits. Removal of the tubulin COOH termini by subtilisin digestion caused all eight CCT subunits to associate with the microtubule polymer, thus highlighting the non-chaperonin nature of the selective CCT subunit association with normal microtubules.Molecular chaperones are a diverse group of proteins that assist the correct folding and intracellular targeting of newly synthesized polypeptides (1) and can modulate the oligomerization and polymerization of folded native proteins (e.g. Ref.2). The chaperonins are a family of molecular chaperones that are characterized by their oligomeric structure, namely a double torus of ϳ60 kDa subunits (3) enclosing a central cavity within which the folding substrate may be sequestered (4 -6). Chaperonin-assisted protein folding proceeds by ATP-driven, alternating cycles of substrate binding and release, ultimately resulting in a native, or near-native, protein that is no longer recognized by the chaperonin (7,8). The cytosolic chaperonin containing T-complex polypeptide 1 (CCT) 1 is the only known chaperonin in the cytosol of eukaryotes (9, 10). The eightmembered rings of the CCT double torus consist usually of eight distinct but related (ϳ30% identity) gene products, CCT␣, -␤, -␥, -␦, -⑀, -, -, and -(11). In yeast, these eight subunits are encoded by essential genes, and mutations in individual subunits lead to defects in the functioning of the cytoskeleton, most commonly manifested as arrest in mitosis (reviewed in Ref. 12). There is both in vivo (10) and in vitro (13-15) evidence that major substrates of the cytosolic chaperonin are tubulins and actins. In addition to assisting folding of newly synthesized tubulins and actins, the CCT␣ subunit appears to be a component of the centrosome and essential for nucleated microtubule assembly from this organelle (16). That this process can take place in a permeabilized cell system, in the absence of protein synthesis, suggests that CCT␣ at least may also be involved in facilitating the polymerization of fully folded tubulins; which, if any, other CCT subunits are required remains to be determined. Indeed, whether CCT subunits always and only exist in cells as c...

Proteomic and Metabolomic Analysis of Vascular Smooth Muscle Cells

“…Besides enzymatic alterations, we observed profound changes in cytoskeletal proteins in PKC␦ Ϫ/Ϫ SMCs, including actin and myosin light chain, which were associated with a compensatory increase in intermediate filaments, eg, vimentin and lamin, and alterations in calcium binding proteins, eg, calmodulin and caldesmon 1 (Table 1). Moreover, PKC␦ deficiency resulted in marked changes of cellular chaperones, including heat shock protein 4 (Hsp4), the tubulin bindingsubunit of the T-complex polypeptide 1 (CCT-1 ), 18 and the redox sensitive chaperone protein disulfide isomerase. 19,20 Further alterations were observed for proteins involved in cell division, eg, septin and immunoregulation, eg, Fkbp9 and annexin 1.…”

Involvement of T-complex Protein-1δ in Dopamine Triggered Apoptosis in Chick Embryo Sympathetic Neurons

“…The role of stress proteins in cell death and survival pathways is an intriguing and controversial issue. Freyaldenhoven and Ali (45) (53) show that ␣,␥, , and subunits can form smaller complexes than those of the CCT complex and act as microtubule-associated proteins. Taken together, these results support the view that CCT subunits can act independently, as TCP-1␦ does in the present study.…”

Revisiting the tubulin folding pathway: new roles in centrosomes and cilia

“…Additionally, CCTs might stabilize microtubule anchoring to MTOCs/caps thus helping these structures to cope, for example, with mechanical stress. In fact, some CCT subunits (a, g, z and u) associate with microtubules polymerized in vitro, behaving as typical MAPs (66). Alternatively, CCTs could be involved in the interactions between microtubules and the ciliary membrane given that cilia caps connect these two systems.…”

“…CCTa and CCTz subunits have been localized at the centrosome throughout the cell cycle in mammalian cells (66,67). In agreement, in the ciliate Tetrahymena pyriformis, the CCTa, CCT´, CCTd and CCTh subunits localize in basal bodies, the oral apparatus, and contractile vacuole pores (68), all structures known to nucleate/organize microtubules (69).…”

“…For example, many CCT interacting proteins contain WD40 domains wfor review see (40)x which are also present in a variety of centrosomal proteins such as the p80 subunit of the microtubule severing protein katanin (71); the centrosomal protein POC1, involved in ciliogenesis and in centriole duplication, stability and length control (72,73); and NEDD1/GCP-WD, a g-tubulin ring complex component (74). CCT subunits, either as free entities or as components of microcomplexes (75), have also been implicated in the regulation of cytoskeleton assembly and dynamics (66,68,76,77), supporting the concept that their function extends beyond the folding assistance or the maintenance of correct tubulin heterodimers concentration.…”