We describe the discovery of a heterohexameric chaperone protein, prefoldin, based on its ability to capture unfolded actin. Prefoldin binds specifically cytosolic chaperonin (c-cpn) and transfers target proteins it. Deletion gene encoding prefoldin subunit in S. cerevisiae results phenotype similar those found when c-cpn is mutated, namely impaired functions actin tubulin-based cytoskeleton. Consistent with having general role chaperonin-mediated folding, we identify homologs archaea, which...

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 neurofibrillary tangles Alzheimer's disease. complete sequence mouse was determined from series overlapping cloned complementary DNAs. carboxyl-terminal 185 amino acids very similar (67 percent) corresponding region protein, and includes three imperfect repeats, each 18 long...

A clone encoding mouse glial fibrillary acidic protein (GFAP) was isolated from a cDNA library constructed so as to express the cloned sequences. The screened using GFAP-specific polyclonal antiserum; single bacterial colony expressing GFAP identified. complete sequence of insert in this is presented, encompassing 2.5 kilobases and specifying greater than 97% amino acid sequence. includes long (1.4-kilobase) 3' untranslated region. Within coding region, data show extensive homology with...

We describe five mouse tubulin cloned cDNAs, two (M alpha 1 and M 2) that encode alpha-tubulin three beta 2, 4, 5) beta-tubulin. The sequence of these clones reveals each represents a distinct gene product. Within the common to encoded amino acids are identical, though 3' noncoding regions wholly dissimilar. In contrast, beta-tubulin cDNAs show considerable carboxy-terminal heterogeneity. Two isotypes defined by sequences absolutely conserved between human, all rat. This result implies...

Five mouse alpha-tubulin isotypes are described, each distinguished by the presence of unique amino acid substitutions within coding region. Most, though not all these isotype-specific acids, clustered at carboxy terminus. One described is expressed exclusively in testis and encoded two closely related genes (M alpha 3 M 7) which have homologous 3' untranslated regions but differ multiple third codon positions their 5' regions. We show that a subfamily encoding same testis-specific isotype...

We describe the structure of a novel and unusually heterologous beta-tubulin isotype (M beta 1) isolated from mouse bone marrow cDNA library, second 3) testis library. Comparison M 1 3 with completed 4, 5) or extended 2) sequence three previously described isotypes shows that each includes distinctive carboxy-terminal region, in addition to multiple amino acid substitutions throughout polypeptide chain. In every case where mammalian interspecies comparison can be made, both internal...

The ADP ribosylation factor-like proteins (Arls) are a family of small monomeric G unknown function. Here, we show that Arl2 interacts with the tubulin-specific chaperone protein known as cofactor D. Cofactors C, D, and E assemble α/β- tubulin heterodimer also interact native tubulin, stimulating it to hydrolyze GTP thus acting together β-tubulin GTPase activating (GAP). We find downregulates GAP activity E, inhibits binding D in vitro. overexpression cofactors or cultured cells results...

The sequence of a human beta-tubulin cDNA clone (D beta-1) is described; our data revealed 95.6% homology compared with the processed pseudogene derived by reverse transcription mRNA (Wilde et al., Nature [London] 297:83-84, 1982). However, amino acid encoded this showed less pig and chicken sequences than latter did to each other, major divergence within 15 carboxy-terminal acids. On other hand, an independently isolated, functionally expressed genomic (5 beta) possessed very high degree...

The production of native α/β tubulin heterodimer in vitro depends on the action cytosolic chaperonin and several protein cofactors. We previously showed that four such cofactors (termed A, C, D, E) together with act β-tubulin folding intermediates generated by to produce polymerizable heterodimers. However, this set generates heterodimers only very inefficiently from α-tubulin produced same chaperonin. Here we describe isolation, characterization, genetic analysis a novel cofactor (cofactor...

A 1.2-kilobase (kb) cDNA clone (NF68) encoding the mouse 68,000-mol-wt neurofilament protein is described. The was isolated from a brain library by low-stringency cross-hybridization with probe glial fibrillary acidic (Lewis et al., 1984, Proc. Natl. Acad. Sci. USA., 81:2743-2746). identity of NF68 established hybrid selection using polyA+ mRNA, and cell-free translation selected mRNA species. product co-migrated authentic on an SDS/polyacrylamide gel, immunoprecipitable monospecific rabbit...

The origin of introns and their role (if any) in gene expression, the evolution genome, generation new expressed sequences are issues that understood poorly, if at all. Multigene families provide a favorable opportunity for examining evolutionary history because it is possible to identify changes intron placement content since divergence family members from common ancestral sequence. Here we report complete sequence encoding 68-kilodalton (kDa) neurofilament protein; member intermediate...

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

In the accompanying paper (Gu, W., S. A. Lewis, and N. J. Cowan. 1988. Cell Biol. 106: 2011-2022), we report generation of three antisera, each which uniquely recognizes a different mammalian alpha-tubulin isotype, plus fourth antibody that distinguishes between microtubules containing tyrosinated nontyrosinated form only known gene product lacks an encoded carboxy-terminal tyrosine residue. These sera, together with five sera raised distinguish among beta-tubulin isotypes, have been used to...

In vivo, many proteins must interact with molecular chaperones to attain their native conformation. the case of tubulin, newly synthesized α- and β-subunits are partially folded by cytosolic chaperonin, a double-toroidal ATPase homologs in all kingdoms life most cellular compartments. β-tubulin folding intermediates then brought together tubulin-specific chaperone (named cofactors A–E) cofactor-containing supercomplex GTPase activity. Here we show that tubulin subunit exchange can only occur...

Abstract The characteristic features of Alzheimer's disease (AD) include a high density β‐amyloid‐containing plaques in the cerebral cortex and loss basal forebrain cholinergic neurons. Amyloid β‐protein (Aβ, Mr. ∼4.5 kDa) is derived from family large (Mr. ∼110–140 β‐amyloid precursor proteins (APP) which are integral membrane glycoproteins consisting extracytoplasmic domain, transmembrane short cytoplasmic tail. Secreted derivatives APP lacking tail, small portion extracellular domain...

Mutations in the X-linked retinitis pigmentosa 2 gene cause progressive degeneration of photoreceptor cells. The protein (RP2) is similar sequence to tubulin-specific chaperone cofactor C. Together with cofactors D and E, C stimulates GTPase activity native tubulin, a reaction regulated by ADP-ribosylation factor-like protein. Here we show that presence D, RP2 also tubulin. We find this function abolished mutation an arginine residue conserved both RP2. Notably, mutations alter codon...

A rapid and sensitive in situ hybridization technique is described for the detection of mRNA sequences 6-8-micron cryostat sections. The method incorporates use alpha-thio-35S-labelled nucleoside triphosphates generation high-specific-activity DNA probes a high-stringency washing procedure that virtually eliminates background without unduly compromising histological integrity. Whereas signal resolution less than observed using 3H probes, 35S-labelled are well-suited experiments where at...

We describe the isolation of a set overlapping cDNAs encoding mouse microtubule associated protein 2 (MAP2), using an anti-MAP antiserum to screen brain cDNA expression library cloned in bacteriophage lambda gt11. The authenticity these clones was established by following criteria: (a) three non-identical each expressing MAP2 immunoreactive fusion were independently isolated from library; cross-hybridized at nucleic acid level; (b) affinity purified nitrocellulose-bound protein; antibodies...