Faculty Bibliography

We have isolated a cytoplasmic chaperonin based on its ability to catalyze the folding of denatured beta-actin. The cytoplasmic chaperonin is organized as a multisubunit toroid and requires Mg2+ and ATP for activity. The folding reaction proceeds via the rapid ATP-independent formation of a binary complex, followed by a slower ATP-dependent release of the native product. Electron microscopic observations reveal a striking structural change that occurs upon addition of Mg2+ and ATP. The eukaryotic cytoplasm thus contains a chaperonin that is functionally analagous to its prokaryotic, mitochondrial, and chloroplastic counterparts

Chaperonins are thought to participate in the process of protein folding in bacteria and in eukaryotic mitochondria and chloroplasts. While some chaperonins are relatively well characterized, the structures of the mammalian chaperonins are unknown. We have expressed a mammalian mitochondrial chaperonin 60 in Escherichia coli and purified the recombinant protein to homogeneity. Structural and biochemical analyses of this protein establish a single toroidal structure of seven subunits, in contrast to the homologous bacterial, fungal, and plant chaperonin 60s, which have double toroidal structures comprising two layers of seven identical subjects each. The recombinant mammalian chaperonin 60, together with the mammalian chaperonin 10 (but not with bacterial chaperonin 10), facilitates the formation of catalytically active ribulose-bisphosphate carboxylase from an unfolded state in the presence of K+ and MgATP. Analysis of the partial reactions involved in this in vitro reconstitution reveals that the single toroid of chaperonin 60 can form stable complexes with both unfolded or partially folded [35S]ribulose-bisphosphate carboxylase and mitochondrial (but not bacterial) chaperonin 10 in the presence of MgATP. We conclude that the minimal functional unit of chaperonin 60 is a single hepatmeric toroid

The functional subunit of microtubules is a heterodimer consisting of alpha- and beta-tubulin. An understanding of tubulin dimerization has been hampered because it has not proved possible to purify native tubulin monomers. To study the process whereby tubulin dimers are formed, we made use of tubulins synthesized by in vitro transcription and translation. We present evidence that the in vitro synthesis of different mouse alpha-tubulin isotypes involves a multimolecular complex. The synthesis of mouse beta-tubulin isotypes also involves the formation of multimolecular complexes, though different isotypes behave somewhat differently from one another. The properties of in vitro synthesized alpha- and beta-tubulin multimolecular complexes strongly suggest that they are intermediates in the biosynthesis of tubulin monomers. Upon release, these monomers can exchange with pre-existing tubulin heterodimers

We show that the expression of the gene encoding glial fibrillary acidic protein (GFAP) gene is affected by at least three cis-acting elements. A positive regulatory element that is located between nucleotides -1,631 and -1,479 can confer cell type-specific expression on a heterologous gene. A second regulatory element is located between nucleotides -97 and -80. The third is a negative regulatory element that is located within the first intron of the gene. Deletion of this element activates GFAP expression in HeLa cells, and affects promoter function in glioma cells.

The regulation of cell type-specific expression of the gene encoding glial filament acidic protein (GFAP) was examined by introducing various deletion mutants of the gene into GFAP-expressing (U251 human astrocytoma) and non-expressing (HeLa) cell lines, and measuring their transcriptional activity in an RNAase protection assay. The expression of GFAP is influenced by a number of cis-acting elements. A domain that resides between nucleotides -1631 and -1479 can confer cell type-specific expression when coupled to a heterologous gene. We also present evidence for the existence of a negative regulatory element that resides within the first intron of the GFAP gene

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 KKEVI 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 KKEVI 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 KKEVI 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

Here we report that the microtubule-associated proteins MAP2 and tau share two separable functional domains. One is the microtubule-binding site which serves to nucleate microtubule assembly; the second is a short C-terminal alpha-helical sequence which can crosslink microtubules by means of a hydrophobic zipper interaction into dense stable parallel arrays characteristic of axons or dendrites. Thus, interactions between molecules of a single type are capable of drastically reorganizing microtubules and completely suppressing their dynamic properties

beta-Tubulin synthesis in eucaryotic cells is subject to control by an autoregulatory posttranscriptional mechanism in which the first four amino acids of the beta-tubulin polypeptide act either directly or indirectly to control the stability of beta-tubulin mRNA. To investigate the contribution of this amino-terminal domain to microtubule assembly and dynamics, we introduced a series of deletions encompassing amino acids 2 to 5 of a single mammalian beta-tubulin isotype, M beta 1. Constructs carrying such deletions were inserted into an expression vector, and the ability of the altered polypeptide to coassemble into microtubules was tested by using an anti-M beta 1-specific antibody. We show that the M beta 1 beta-tubulin polypeptide was competent for coassembly into microtubules in transient transfection experiments and in stably transfected cell lines when it lacked either amino acid 2 or amino acids 2 and 3. The capacity of these mutant beta-tubulins to coassemble into polymerized microtubules was only slightly diminished relative to that of unaltered beta-tubulin, and their expression did not influence the viability or growth properties of cell lines carrying these deletions. However, more extensive amino-terminal deletions either severely compromised or abolished the capacity for coassembly. In analogous experiments in which alterations were introduced into the amino-terminal domain of a mammalian alpha-tubulin isotype, M alpha 4, deletion of amino acid 2 did not affect the ability of the altered polypeptide to coassemble, although removal of additional amino-terminal residues essentially abolished the capacity for competent coassembly. The stability of the altered assembly-competent alpha- and beta-tubulin polypeptides was measured in pulse-chase experiments and found to be indistinguishable from the stability of the corresponding unaltered polypeptides. An assembly-competent M alpha 4 polypeptide carrying a deletion encompassing the 12 carboxy-terminal amino acids also had a half-life indistinguishable from that of the wild-type alpha-tubulin molecule. These data suggest that the universally conserved amino terminus of beta-tubulin acts largely in a regulatory role and that the carboxy-terminal domain of alpha-tubulin is not essential for coassembly in mammalian cells in vivo