Slow axonal transport of the cytosolic chaperonin CCT with Hsc73 and actin in motor neurons

Abstract

Molecular chaperones are well known for their role in facilitating the folding of nascent and newly synthesized proteins, but have other roles, including the assembly, translocation and renaturation of intracellular proteins. Axons are convenient tissues for the study of some of these other roles because they lack the capacity for significant protein synthesis. We examine the axonal transport of the cytosolic chaperonin containing T- complex polypeptide 1 (CCT) by labeling lumbar motor neurons with [35S]methionine and examining sciatic nerve proteins by 2-D gel electrophoresis and immunoblotting. All CCT subunits identifiable with specific antibodies, namely CCTalpha, CCTbeta, CCTgamma and CCTepsilon/CCTtheta; (the latter two subunits colocalized in analyses of rat nerve samples), appeared to be labeled in "slow component b" of axonal transport along with the molecular chaperone Hsc73 and actin, a major folding substrate for CCT. Our results are consistent with molecular chaperones having a post-translational role in maintaining the native form of actin during its slow transport to the axon terminal and ensuring its correct assembly into microfilaments.

Mutation in the epsilon subunit of the cytosolic chaperonin-containing t-complex peptide-1 (Cct5) gene causes autosomal recessive mutilating sensory neuropathy with spastic paraplegia

“…However, since it has been reported that the equatorial domain could be involved in ATP binding and the apical domain in substrate binding, 16 the mutation may affect the binding and hydrolysis of ATP, affecting the epsilon subunit ability to bind and fold its specific substrates, mainly actin and tubulin, the two major proteins folded by the CCT complex. Misfolding of these cytoskeleton proteins by the defect in the ATP binding and folding activity of the CCT epsilon subunit may cause the defect in their slow axonal transport and their assembly into microfilaments as reported by Bourke et al, 18 leading to apoptosis. This mechanism is in accordance with the findings that in all patients of the IDR family, the sensory neuropathy was distal with ulcerations starting in toes and fingers, and neuronal apoptosis in the spinal cord.…”

Off-Target Effects of Psychoactive Drugs Revealed by Genome-Wide Assays in Yeast

“…The human homolog of this gene is associated with hereditary neuropathy [40]. Although it is unclear how mutated CCT5 causes this disease, it has been postulated that its mutation leads to accumulation of misfolded cytoskeletal proteins, leading to defective assembly of actin into microfilaments resulting in neuronal apoptosis [41]. In our yeast screens, CCT5 was needed for resistance to eight different compounds (cyproheptadine, paroxetine, fluoxetine, indatraline, MDL72222, CY208-243, 2-Chloro-11-(4-methylpiperazino)-dibenz[b,f]oxepin, N-Desmethyl-clozapine, and 3-alpha-[(4-Chlorophenyl)-phenylmethoxy]-tropane.…”

Cytoskeletal Requirements in Axonal Transport of Slow Component-b

“…SCb moves at average rates of 2-8 mm/d and transports Ϸ200 proteins, of which only a handful have been identified. Among these are myosin, dynein, clathrin, calmodulin, synapsin, ␣-synuclein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), superoxide dismutase-1, phosphofructokinase, ubiquitin, molecular chaperones, tau and other microtubule-associated proteins (MAPs), spectrin, actindepolymerizing factor, and actin (Willard et al, 1974;Black and Lasek, 1979;Brady et al, 1981;Garner and Lasek, 1982;Lasek et al, 1984;Baitinger and Willard, 1987;Nixon et al, 1990;Bray et al, 1992;Mercken et al, 1995;Dillman et al, 1996;Yuan et al, 1999;Ma et al, 2000;Bourke et al, 2002;Li et al, 2004). Clearly, the proteins of SCb are functionally diverse, play critical roles in maintaining the integrity of axons and synapses, and are implicated in neurodegenerative diseases .…”