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.

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 Alzheimer's 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.

Abstract. We report the complete sequence of the microtubule-associated protein MAP1B, deduced from a series of overlapping genomic and cDNA clones. The encoded protein has a predicted molecular mass of 255,534 D and contains two unusual sequences. The first is a highly basic region that includes multiple copies of a short motif of the form KKEE or KKF_~ that are repeated, but not at exact intervals. The second is a set of 12 imperfect repeats, each of 15 amino acids and each spaced by two amino acids. Subcloned fragments spanning these two distinctive regions were expressed as labeled polypeptides by translation in a cell-free system in vitro. These polypeptides were tested for their ability to copurify with unlabeled brain microtubules through successive cycles of polymerization and depolymerization. The peptide corresponding to the region containing the KKEE and KKE~ motifs cycled with brain microtubules, whereas the peptide corresponding to the set of 12 imperfect repeats did not. To define the microtubule binding domain in vivo, full-length and deletion constructs encoding MAP1B were assembled and introduced into cultured cells by transfection. The expression of transfected polypeptides was monitored by indirect immunofluorescence using anti-MAP1B-specific antisera. These experiments showed that the basic region containing the KKEE and KKF_~ motifs is responsible for the interaction between MAP1B and microtubules in vivo. This region bears no sequence relationship to the microtubule binding domains of kinesin, MAP2, or tau.

The ADP ribosylation factor-like proteins (Arls) are a family of small monomeric G proteins of unknown function. Here, we show that Arl2 interacts with the tubulin-specific chaperone protein known as cofactor D. Cofactors C, D, and E assemble the α/β- tubulin heterodimer and also interact with native tubulin, stimulating it to hydrolyze GTP and thus acting together as a β-tubulin GTPase activating protein (GAP). We find that Arl2 downregulates the tubulin GAP activity of C, D, and E, and inhibits the binding of D to native tubulin in vitro. We also find that overexpression of cofactors D or E in cultured cells results in the destruction of the tubulin heterodimer and of microtubules. Arl2 specifically prevents destruction of tubulin and microtubules by cofactor D, but not by cofactor E. We generated mutant forms of Arl2 based on the known properties of classical Ras-family mutations. Experiments using these altered forms of Arl2 in vitro and in vivo demonstrate that it is GDP-bound Arl2 that interacts with cofactor D, thereby averting tubulin and microtubule destruction. These data establish a role for Arl2 in modulating the interaction of tubulin-folding cofactors with native tubulin in vivo.

The production of native α/β tubulin heterodimer in vitro depends on the action of cytosolic chaperonin and several protein cofactors. We previously showed that four such cofactors (termed A, C, D, and E) together with native tubulin act on β-tubulin folding intermediates generated by the chaperonin to produce polymerizable tubulin heterodimers. However, this set of cofactors generates native heterodimers only very inefficiently from α-tubulin folding intermediates produced by the same chaperonin. Here we describe the isolation, characterization, and genetic analysis of a novel tubulin folding cofactor (cofactor B) that greatly enhances the efficiency of α-tubulin folding in vitro. This enabled an integrated study of α- and β-tubulin folding: we find that the pathways leading to the formation of native α- and β-tubulin converge in that the folding of the α subunit requires the participation of cofactor complexes containing the β subunit and vice versa. We also show that sequestration of native α-or β-tubulins by complex formation with cofactors results in the destabilization and decay of the remaining free subunit. These data demonstrate that tubulin folding cofactors function by placing and/or maintaining α-and β-tubulin polypeptides in an activated conformational state required for the formation of native α/β heterodimers, and imply that each subunit provides information necessary for the proper folding of the other.

We describe five mouse tubulin cloned cDNAs, two (Mal and Ma2) that encode a-tubulin and three (M/32, M/34, and Mfl5) that encode /3-tubulin. The sequence of these clones reveals that each represents a distinct gene product. Within the sequence common to the two a-tubulin cDNAs, the encoded amino acids are identical, though the 3' noncoding regions are wholly dissimilar. In contrast, the three /~-tubulin cDNAs show considerable carboxy-terminal heterogeneity. Two of the /3-tubulin isotypes defined by the cloned sequences are absolutely conserved between mouse and human, and all three/3-tubulin isotypes are conserved between mouse and rat. This result implies the existence of selective constraints that have maintained sequence identity after species divergence. This conclusion is reinforced by the near identity between a third mouse ~-tubulin isotype and a chicken/3-tubulin isotype.

contributed equally to this workThe 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 speci®c CCT subunits that are placed in a 1,4 arrangement. This de®nes the phasing of the CCT rings and suggests a handoff mechanism for PFD.

A clone encoding mouse glial fibrillary acidic protein (GFAP) was isolated from a cDNA library constructed so as to express the cloned sequences. The library was screened using a GFAP-specific polyclonal antiserum; a single bacterial colony expressing GFAP was identified. The complete sequence of the cDNA insert in this clone is presented, encompassing 2.5 kilobases and specifying >97% of the GFAP amino acid sequence. The clone includes a long (1.4-kilobase) (GFAP). This protein is of particular interest because it represents a specific marker in the development of the central nervous system, its presence distinguishing astrocytes from other glial cell types. Here we describe the isolation and complete sequence of a 2.5-kilobase (kb) cDNA clone encoding mouse GFAP. The structural and evolutionary implications of the predicted amino acid sequence of mouse GFAP are discussed.