Sally A. Lewis

Prefoldin, a Chaperone that Delivers Unfolded Proteins to Cytosolic Chaperonin

We describe the discovery of a heterohexameric chaperone protein, prefoldin, based on its ability to capture unfolded actin. Prefoldin binds specifically to cytosolic chaperonin (c-cpn) and transfers target proteins to it. Deletion of the gene encoding a prefoldin subunit in S. cerevisiae results in a phenotype similar to those found when c-cpn is mutated, namely impaired functions of the actin and tubulin-based cytoskeleton. Consistent with prefoldin having a general role in chaperonin-mediated folding, we identify homologs in archaea, which have a class II chaperonin but contain neither actin nor tubulin. We show that by directing target proteins to chaperonin, prefoldin promotes folding in an environment in which there are many competing pathways for nonnative proteins.

Microtubule-associated protein MAP2 shares a microtubule binding motif with tau protein

The microtubule-associated protein MAP2 is a prominent large-sized component of purified brain microtubules that, like the 36- to 38-kilodalton tau proteins, bears antigenic determinants found in association with the neurofibrillary tangles of Alzheimers disease. The complete sequence of mouse brain MAP2 was determined from a series of overlapping cloned complementary DNAs. The sequence of the carboxyl-terminal 185 amino acids is very similar (67 percent) to a corresponding region of tau protein, and includes a series of three imperfect repeats, each 18 amino acids long and separated by 13 or 14 amino acids. A subcloned fragment spanning the first two of the 18-amino acid repeats was expressed as a polypeptide by translation in vitro. This polypeptide copurified with microtubules through two successive cycles of polymerization and depolymerization, whereas a control polypeptide derived from the amino-terminal region of MAP2 completely failed to copurify. These data imply that the carboxyl-terminal domain containing the 18-amino acid repeats constitutes the microtubule binding site in MAP2. The occurrence of these repeats in tau protein suggests that these may be a general feature of microtubule binding proteins.

Free intermingling of mammalian β-tubulin isotypes among functionally distinct microtubules

Mammalian cells express a spectrum of tubulin isotypes whose relationship to the diversity of microtubule function is unknown. To examine whether different isotypes are segregated into functionally distinct microtubules, we generated immune sera capable of discriminating among the various naturally occurring beta-tubulin isotypes. Cloned fusion proteins encoding each isotype were used first to tolerogenize animals against shared epitopes, and then as immunogens to elicit a specific response. In experiments using these sera, we show that there is neither complete nor partial segregation of beta-tubulin isotypes: both interphase cytoskeletal and mitotic spindle microtubules are mixed copolymers of all expressed beta-tubulin isotypes. Indeed, a highly divergent isotype normally expressed only in certain hematopoietic cells is also indiscriminately assembled into all microtubules both in their normal context and when transfected into HeLa cells.

Structure of eukaryotic prefoldin and of its complexes with unfolded actin and the cytosolic chaperonin CCT

The biogenesis of the cytoskeletal proteins actin and tubulin involves interaction of nascent chains of each of the two proteins with the oligomeric protein prefoldin (PFD) and their subsequent transfer to the cytosolic chaperonin CCT (chaperonin containing TCP‐1). Here we show by electron microscopy that eukaryotic PFD, which has a similar structure to its archaeal counterpart, interacts with unfolded actin along the tips of its projecting arms. In its PFD‐bound state, actin seems to acquire a conformation similar to that adopted when it is bound to CCT. Three‐dimensional reconstruction of the CCT:PFD complex based on cryoelectron microscopy reveals that PFD binds to each of the CCT rings in a unique conformation through two specific CCT subunits that are placed in a 1,4 arrangement. This defines the phasing of the CCT rings and suggests a handoff mechanism for PFD.

Differential distribution of beta-tubulin isotypes in cerebellum.

We describe the structure and expression of a mammalian beta‐tubulin isotype (M beta 6) that is weakly expressed in testis but is abundant in developing brain, with transcripts declining to lower levels in the adult brain. The expression of M beta 6 was undetectable in any other mouse tissue examined. A serum specific for this isotype was prepared using a cloned fusion protein as immunogen. M beta 6 is one of five known beta‐tubulin isotypes expressed in brain, and using the anti‐M beta 6 serum along with sera, anti‐M beta 2, anti‐M beta 3/4 and anti‐M beta 5, previously characterized, we have examined the pattern of expression of beta‐tubulin isotypes in rat cerebellum. The isotypes each have characteristic cell‐type specific patterns of localization in cerebellum. M beta 2, M beta 3/4 and M beta 5 are present in both neuronal and non‐neuronal cells, but in contrast M beta 6 was only detectable in neurons in tissue sections and in dissociated cerebellar cell culture. The majority of sequence differences among the beta‐tubulin isotypes lie at the carboxy terminus, the region of beta‐tubulin involved in MAP binding. In the case of M beta 2 and M beta 6, the patterns of expression are similar or identical to the patterns of expression of MAP3 and MAP1A respectively. These results suggest that beta‐tubulin isotypes may contribute to the determination of the specific association of MAPs with microtubules of diverse function. However, the strict subcellular segregation of other MAPs in brain may be determined by other factors.

The α- and β-tubulin folding pathways

The α—β tubulin heterodimer is the subunit from which microtubules are assembled. The pathway leading to correctly folded α- and β-tubulins is unusually complex: it involves cycles of ATP-dependent interaction of newly synthesized tubulin subunits with cytosolic chaperonin, resulting in the production of quasi-native folding intermediates, which must then be acted upon by additional protein cofactors. These cofactors form a supercomplex containing both α- and β-tubulin polypeptides, from which native heterodimer is released in a GTP-dependent reaction. Here, we discuss the current state of our understanding of the function of cytosolic chaperonin and cofactors in tubulin folding.

Tubulin folding cofactors as GTPase-activating proteins. GTP hydrolysis and the assembly of the alpha/beta-tubulin heterodimer.

In vivo, many proteins must interact with molecular chaperones to attain their native conformation. In the case of tubulin, newly synthesized α- and β-subunits are partially folded by cytosolic chaperonin, a double-toroidal ATPase with homologs in all kingdoms of life and in most cellular compartments. α- and β-tubulin folding intermediates are then brought together by tubulin-specific chaperone proteins (named cofactors A–E) in a cofactor-containing supercomplex with GTPase activity. Here we show that tubulin subunit exchange can only occur by passage through this supercomplex, thus defining it as a dimer-making machine. We also show that hydrolysis of GTP by β-tubulin in the supercomplex acts as a switch for the release of native tubulin heterodimer. In this folding reaction and in the related reaction of tubulin-folding cofactors with native tubulin, the cofactors behave as GTPase-activating proteins, stimulating the GTP-binding protein β-tubulin to hydrolyze its GTP.

Temporal Expression of Mouse Glial Fibrillary Acidic Protein mRNA Studied by a Rapid In Situ Hybridization Procedure

Abstract: A rapid and sensitive in situ hybridization technique is described for the detection of mRNA sequences in 6–8‐μm cryostat sections. The method incorporates the use of α‐thio‐35S‐labelled nucleoside triphosphates for the generation of high‐specific‐activity DNA probes and a high‐stringency washing procedure that virtually eliminates background without unduly compromising histological integrity. Whereas signal resolution is less than that observed using 3H probes, 35S‐labelled probes are well‐suited for experiments where resolution at the cellular level is required. The method has been applied to a study of the developmental regulation of glial fibrillary acidic protein (GFAP) mRNA expression in developing mouse brain. GFAP‐specific sequences are first detectable after the second postnatal day, and thereafter rise to a level that is maintained throughout development and into adulthood. The distribution of GFAP‐encoding sequences broadly reflects the known distribution of astrocytes, but the levels of mRNA within these cells vary by a surprisingly large amount depending on their location. For example, in adult animals, the astrocytes of the glial limitans contain an abundance of GFAP‐specific mRNA that is higher than corresponding levels in astrocytes in the cerebellar white matter, whereas these cells in turn contain considerably more GFAP‐specific mRNA than astrocytes in the gray matter of the cerebrum. Unexpectedly, parallel RNA blot transfer experiments show the existence of some GFAP‐encoding mRNA size heterogeneity that is restricted to the first postnatal week.

Functional overlap between retinitis pigmentosa 2 protein and the tubulin-specific chaperone cofactor C

Mutations in the X-linked retinitis pigmentosa 2 gene cause progressive degeneration of photoreceptor cells. The retinitis pigmentosa 2 protein (RP2) is similar in sequence to the tubulin-specific chaperone cofactor C. Together with cofactors D and E, cofactor C stimulates the GTPase activity of native tubulin, a reaction regulated by ADP-ribosylation factor-like 2 protein. Here we show that in the presence of cofactor D, RP2 protein also stimulates the GTPase activity of tubulin. We find that this function is abolished by mutation in an arginine residue that is conserved in both cofactor C and RP2. Notably, mutations that alter this arginine codon cause familial retinitis pigmentosa. Our data imply that this residue acts as an “arginine finger” to trigger the tubulin GTPase activity and suggest that loss of this function in RP2 contributes to retinal degeneration. We also show that in Saccharomyces cerevisiae, both cofactor C and RP2 partially complement the microtubule phenotype resulting from deletion of the cofactor C homolog, demonstrating their functional overlap in vivo. Finally, we find that RP2 interacts with GTP-bound ADP ribosylation factor-like 3 protein, providing a link between RP2 and several retinal-specific proteins, mutations in which also cause retinitis pigmentosa.

Quasi-native Chaperonin-bound Intermediates in Facilitated Protein Folding

Chaperonins are known to facilitate protein folding, but their mechanism of action is not well understood. The fact that target proteins are released from and rebind to different chaperonin molecules (“cycling”) during a folding reaction suggests that chaperonins function by unfolding aberrantly folded molecules, allowing them multiple opportunities to reach the native state in bulk solution. Here we show that the cycling of α-tubulin by cytosolic chaperonin (c-cpn) can be uncoupled from the action of cofactors required to complete the folding reaction. This results in the accumulation of folding intermediates which are chaperonin-bound, stable, and quasi-native in that they bind GTP nonexchangeably. We present evidence that these intermediates can be generated without the target protein leaving c-cpn. These data show that, in contrast to prevailing models, target proteins can maintain, and possibly acquire, significant native-like structure while chaperonin-bound.