A dynactin subunit with a highly conserved cysteine-rich motif interacts directly with Arp1.

Dynactin is a multisubunit complex and a required cofactor for most, or all, of the cellular processes powered by the microtubule-based motor cytoplasmic dynein. Using a dynein affinity column, the previously uncharacterized p62 subunit of dynactin was isolated and microsequenced. Two peptide sequences were used to clone human cDNAs encoding p62. Sequence analysis of the predicted human polypeptide of 53 kDa revealed a highly conserved pattern of eleven cysteine residues, eight of which fit the consensus sequence for a Zn(2+)-binding RING domain. We have characterized p62 as an integral component of 20 S dynactin by biochemical and immunocytochemical methods. Affinity chromatography experiments demonstrate that p62 binds directly to the Arp1 subunit of dynactin. Immunocytochemistry with antibodies to p62 demonstrates that this polypeptide has a punctate cytoplasmic distribution as well as centrosomal distribution typical of dynactin. In transfected cells, overexpression of p62 did not disrupt microtubule organization or the integrity of the Golgi but did cause both cytosolic and nuclear distribution of the protein, suggesting that this polypeptide may be targeted to the nucleus at very high expression levels.

The genomic structure of DCTN1, a candidate gene for limb-girdle muscular dystrophy (LGMD2B).

Dynactin is a required activator for the molecular motor cytoplasmic dynein, and is likely to be essential for normal neuronal development. Previously we mapped the human gene encoding the p150Glued subunit of dynactin to 2p13, in the vicinity of the locus linked to limb-girdle muscular dystrophy (LGMB2B). We now report the genomic organization of DCTN1. We have identified 32 exons in the gene which spans approximately 25 kb. Alternative splicing of several of the exons generates functionally distinct isoforms of the p150Glued polypeptide.

Interactions between the cytochrome b, cytochrome c1, and Fe-S protein subunits at the ubihydroquinone oxidation site of the bc1 complex of Rhodobacter capsulatus.

Ubihydroquinone:cytochrome (cyt) c oxidoreductase (bc1 complex and its plant counterpart b6f complex) is a vital component of energy-transducing systems in most organisms from bacteria to eukaryotes. In the facultative phototrophic (Ps) bacterium Rhodobacter capsulatus, it is constituted by the cyt b, cyt c1, and Rieske Fe-S protein subunits and is essential for Ps growth. Of these subunits, cyt b has two nontransmembrane helices, cd1 and cd2, which are critical for its structure and function. In particular, substitution of threonine (T) at position 163 on cd1 with phenylalanine (F) or proline (P) leads to the absence of the bc1 complex. Here, Ps+ revertants of B:T163F were obtained, and their detailed characterizations indicated that position 163 is important for the assembly of the bc1 complex by mediating subunit interactions at the Qo site. The loss of the hydroxyl group at position 163 of cyt b was compensated for by the gain of either a hydroxyl group at position 182 of cyt b or 46 of the Fe-S protein or a sulfhydryl group at position 46 of cyt c1. Examination of the mitochondrial bc1 complex crystal structure [Zhang, Z., Huang, L., Shulmeister, V. M., Chi, Y.-I., Kim, K. K., Hung, L.-W., Crofts, A. R., Berry, E. A., and Kim, S.-H. (1998) Nature 392, 677-684] revealed that the counterparts of B:G182 (i.e., G167) and F:A46 (i.e. , A70) are located close to B:T163 (i.e., T148), whereas the C:R46 (i.e., R28) is remarkably far from it. The revertants contained substoichiometric amounts of the Fe-S protein subunit and exhibited steady-state and single-turnover, electron transfer activities lower than that of a wild-type bc1 complex. Interestingly, their membrane supernatants contained a smaller form of this subunit with physicochemical properties identical to those of its membrane-bound form. Determination of the amino-terminal amino acid sequence of this soluble Fe-S protein revealed that it was derived from the wild-type protein by proteolytic cleavage at V44. This work revealed for the first time that position 163 of cyt b is important both for proper subunit interactions at the Qo site and for inactivation of the bc1 complex by proteolytic cleavage of its Fe-S protein subunit at a region apparently responsible for its mobility during Qo site catalysis.

Casein kinase II binds to and phosphorylates cytoplasmic dynein.

We have isolated a 27-kDa protein that binds to cytoplasmic dynein. Microsequencing of a 17-amino acid peptide of this polypeptide yielded a sequence which completely matched the predicted sequence of the beta subunit of casein kinase II, a highly conserved serine/threonine kinase. Affinity chromatography using a dynein column indicates that both the alpha and beta subunits of casein kinase II are retained by the column from rat brain cytosol. Although dynactin is also bound to the column, casein kinase II is not a dynactin subunit. Casein kinase II does not co-immunoprecipitate with dynactin, and it binds to a dynein intermediate chain column which has been preblocked with excess p150(Glued), a treatment that inhibits the binding of dynactin from cytosol. Bacterially expressed and purified rat dynein intermediate chain can be phosphorylated by casein kinase II in vitro. Further, native cytoplasmic dynein purified from rat brain can also be phosphorylated by casein kinase II in vitro. We propose that CKII may be involved in the regulation of dynein function possibly by altering its cargo specificity or its ability to interact with dynactin.

Mouse p150Glued (dynactin 1) cDNA sequence and evaluation as a candidate for the neuromuscular disease mutation mnd2.

p150Glued (Dynactin 1) is a component of the dynactin complex that is essential for retrograde axonal transport in neurons. The mouse p150Glued cDNA was isolated from mouse brain by RT-PCR. The complete sequence of the full length mouse cDNA was determined, including 19 bp of 5' UTR, 3843 bp of coding sequence, and 162 bp of 3' UTR. The predicted protein has a molecular mass of 142 kDa and exhibits 95% amino acid sequence identity to the predicted amino acid sequence of human p150Glued (DCTN1). The mouse Dctn1 gene was previously mapped in the central region of mouse chromosome 6, close to the neuromuscular disease gene mnd2. Northern blot analysis, complete sequencing of the cDNA, Western blot, and functional tests of the protein did not detect any abnormalities of p150Glued in mnd2 mice.

Huntingtin-associated protein 1 (HAP1) interacts with the p150Glued subunit of dynactin.

Huntington's disease (HD) is an inherited neurodegenerative disease caused by expansion of a polyglutamine repeat in the HD protein huntingtin. Huntingtin's localization within the cell includes an association with cytoskeletal elements and vesicles. We previously identified a protein (HAP1) which binds to huntingtin in a glutamine repeat length-dependent manner. We now report that HAP1 interacts with cytoskeletal proteins, namely the p150 Glued subunit of dynactin and the pericentriolar protein PCM-1. Structural predictions indicate that both HAP1 and the interacting proteins have a high probability of forming coiled coils. We examined the interaction of HAP1 with p150 Glued . Binding of HAP1 to p150 Glued (amino acids 879-1150) was confirmed in vitro by binding of p150 Glued to a HAP1-GST fusion protein immobilized on glutathione-Sepharose beads. Also, HAP1 co-immunoprecipitated with p150 Glued from brain extracts, indicating that the interaction occurs in vivo . Like HAP1, p150 Glued is highly expressed in neurons in brain and both proteins are enriched in a nerve terminal vesicle-rich fraction. Double label immunofluorescence experiments in NGF-treated PC12 cells using confocal microscopy revealed that HAP1 and p150 Glued partially co-localize. These results suggest that HAP1 might function as an adaptor protein using coiled coils to mediate interactions among cytoskeletal, vesicular and motor proteins. Thus, HAP1 and huntingtin may play a role in vesicle trafficking within the cell and disruption of this function could contribute to the neuronal dysfunction and death seen in HD.

Centractin (ARP1) associates with spectrin revealing a potential mechanism to link dynactin to intracellular organelles.

Centractin (Arp1), an actin-related protein, is a component of the dynactin complex. To investigate potential functions of the protein, we used transient transfections to overexpress centractin in mammalian cells. We observed that the overexpressed polypeptide formed filamentous structures that were significantly longer and more variable in length than those observed in the native dynactin complex. The centractin filaments were distinct from conventional actin in subunit composition and pharmacology as demonstrated by the absence of immunoreactivity of these filaments with an actin-specific antibody, by resistance to treatment with the drug cytochalasin D, and by the inability to bind phalloidin. We examined the transfected cells for evidence of specific associations of the novel centractin filaments with cellular organelles or cytoskeletal proteins. Using immunocytochemistry we observed the colocalization of Golgi marker proteins with the centractin polymers. Additional immunocytochemical analysis using antibodies to non-erythroid spectrin (fodrin) and Golgi-spectrin (beta I sigma *) revealed that spectrin colocalized with the centractin filaments in transfected cells. Biochemical assays demonstrated that spectrin was present in dynactin-enriched cellular fractions, was coimmunoprecipitated from rat brain cytosol using antibodies to dynactin subunits, and was coeluted with dynactin using affinity chromatography. Immunoprecipitations and affinity chromatography also revealed that actin is not a bona fide component of dynactin. Our results indicate that spectrin is associated with the dynactin complex. We suggest a model in which dynactin associates with the Golgi through an interaction between the centractin filament of the dynactin complex and a spectrin-linked cytoskeletal network.

Functionally distinct isoforms of dynactin are expressed in human neurons.

P150Glued is the largest subunit of dynactin, which binds to cytoplasmic dynein and activates vesicle transport along microtubules. We have isolated human cDNAs encoding p150Glued as well as a 135-kDa isoform; these isoforms are expressed in human brain by alternative mRNA splicing of the human DCTN1 gene. The p135 isoform lacks the consensus microtubule-binding motif shared by members of the p150Glued/Glued/CLIP-170/BIK1 family of microtubule-associated proteins and, therefore, is predicted not to bind directly to microtubules. We used transient transfection assays and in vitro microtubule-binding assays to demonstrate that the p150 isoform binds to microtubules, but the p135 isoform does not. However, both isoforms bind to cytoplasmic dynein, and both partition similarly into cytosolic and membrane cellular fractions. Sequential immunoprecipitations with an isoform-specific antibody for p150 followed by a pan-isoform antibody revealed that, in brain, these polypeptides assemble to form distinct complexes, each of which sediments at approximately 20 S. On the basis of these observations, we hypothesize that there is a conserved neuronal function for a distinct form of the dynactin complex that cannot bind directly to cellular microtubules.

Ubiquinone pair in the Qo site central to the primary energy conversion reactions of cytochrome bc1 complex.

The mechanistic heart of the ubihydroquinone-cytochrome c oxidoreductase (cyt bc1 complex) is the catalytic oxidation of ubihydroquinone (QH2) at the Qo site. QH2 oxidation is initiated by ferri-cyt c, mediated by the cyt c1 and [2Fe-2S] cluster of the cytochrome bc1 complex. QH2 oxidation in turn drives transmembrane electronic charge separation through two b-type hemes to another ubiquinone (Q) at the Qi site. In earlier studies, residues F144 and G158 of the b-heme containing polypeptide of the Rhodobacter capsulatus cyt bc1 complex were shown to be influential in Qo site function. In the present study, F144 and G158 have each been singly substituted by neutral residues and the dissociation constants measured for both Q and QH2 at each of the strong and weak binding Qo site domains (Qos and Qow). Various substitutions at F144 or G158 were found to weaken the affinities for Q and QH2 at both the Qos and Qow domains variably from zero to beyond 10(3)-fold. This produced a family of Qo sites with Qos and Qow domain occupancies ranging from nearly full to nearly empty at the prevailing approximately 3 x 10(-2) M concentration of the membrane ubiquinone pool (Qpool). In each mutant, the affinity of the Qos domain remained typically 10-20-fold higher than that of the Qow domain, as is found for wild type, thereby indicating that the single mutations caused comparable extents of the weakening at each domain. Moreover, the substitutions were found to cause similar decreases of the affinities of both Q and QH2 in each domain, thereby maintaining the Q/QH2 redox midpoint potentials (Em7) of the Qo site at values similar to that of the wild type. Measurement of the yield and rate of QH2 oxidation generated by single turnover flashes in the family of mutants suggests that the Qos and Qow domains serve different roles for the catalytic process. The yield of the QH2 oxidation correlates linearly with Qos domain occupancy (QH2 or Q), suggesting that the Qos domain exchanges Q or QH2 with the Qpool at a rate which is much slower than the time scale of turnover.(ABSTRACT TRUNCATED AT 400 WORDS)

Expression of the p150Glued component of the dynactin complex in developing and adult rat brain.

p150Glued is a component of the dynactin (Glued) complex that has been shown in vitro to be a required activator of cytoplasmic dynein-mediated transport of vesicles along microtubules and, thus, may be an essential component of retrograde axonal transport. In vivo, a dominant mutation in the Drosophila homologue of p150Glued induces aberrant neuronal development when heterozygous and is lethal when homozygous. In order to characterize the role of the dynactin complex in the development and function of vertebrate neurons, the distribution of the p150Glued message was examined via in situ hybridization to serial sections of adult rat brain and to a developmental series of sections. In the adult rat brain, the most intense hybridization observed with the p150Glued probe was in the pyramidal cells of the hippocampus proper, the dentate granule neurons, the cingulate and piriform cortices, the ventromedial hypothalamus, and the granular cell layer of the cerebellum. White-matter fiber tracts and the neuropil were generally devoid of signal. The data indicate that the mRNA encoding p150Glued is highly enriched in the cell bodies of neurons within the central nervous system. In developing rat, p150Glued is expressed at very high levels in neural tissue from the earliest time points assayed. Particularly intense hybridization was observed in the multiple layers of the retina, which is consistent with the phenotype of the Drosophila mutation. Therefore, the distributions observed via in situ hybridization are consistent with an essential role for p150Glued in retrograde axonal transport.

Roles in inhibitor recognition and quinol oxidation of the amino acid side chains at positions of cyt b providing resistance to Qo-inhibitors of the bc1 complex from Rhodobacter capsulatus.

The substitutions M1401, F144S and L, G152S, T163A and V333A in cytochrome b of the ubiquinol-cytochrome c oxidoreductase (bc1 complex) from Rhodobacter capsulatus provide resistance to the quinol oxidation (Qo) inhibitors myxothiazol, mucidin and stigmatellin. Site-directed mutagenesis with degenerate primers was used to define the role of these positions in inhibitor recognition and quinol oxidation, and a collection of various substitutions at each of these positions was obtained. The effects of these mutations on quinol oxidation, nature and level of inhibitor resistance, prosthetic group incorporation and assembly of the complex were analysed. Most of these mutations, unlike those at position 158 reported earlier, yielded functional bc1 complexes able to support the photosynthetic growth of R. capsulatus. However, they perturbed steady-state quinol oxidation and inhibitor recognition indicating that they are important for the function of the Qo site. In particular, the presence of a methyl group on the beta-carbon (Ile and Val residues) at position 140, the absence of an aromatic ring (Phe, Tyr and Trp residues) at position 144 and the loss of residues with small side chains (Gly and Ala) at position 152 correlated with resistance to myxothiazol. On the other hand, no myxothiazol resistance was observed with the substitutions at positions 163 and 333 suggesting that they affected solely the recognition of stigmatellin. Five substitutions, M140R, F144H and R, G152P and T163R, yielded photosynthesis-deficient mutants with assembled but impaired bc1 complexes. Unexpectedly, two substitutions at position 163 (T to F or P) yielded mutants lacking the three subunits of the bc1 complex indicating that this position affects its assembly or stability in vivo. These findings are discussed in terms of the contributions of these residues to inhibitor recognition and quinol oxidation at the Qo site of the bc1 complex.

petR, located upstream of the fbcFBC operon encoding the cytochrome bc1 complex, is homologous to bacterial response regulators and necessary for photosynthetic and respiratory growth of Rhodobacter capsulatus.

Interposon mutagenesis of a region upstream of the petABC(fbcFBC) operon, encoding the ubiquinol: cytochrome c2 oxidoreductase (bc1 complex) of the photosynthetic bacterium Rhodobacter capsulatus revealed the presence of two genes, petP and petR. DNA nucleotide sequence determination of this region indicated that petP and petR are transcribed in the same direction as the petABC(fbcFBC) operon, and are translationally coupled. A silent insertion located in the interoperonal region separating petPR and the petABC(fbcFBC) genes indicated that these clusters have separate promoters. The deduced amino acid sequence of the putative petR gene product is homologous to various bacterial response regulators, especially to those of the OmpR subgroup. Moreover, it was found that PetR mutants are unable to grow on rich or minimal media by either photosynthesis or respiration, demonstrating that these gene products are essential for growth of R. capsulatus.

Characterization of the pet operon of Rhodospirillum rubrum.

The three genes of the pet operon, coding, respectively, for the Rieske iron-sulfur protein, cytochrome b and cytochrome c 1 components of the cytochrome bc 1 complex in the photosynthetic bacterium Rhodospirillum rubrum have been sequenced. The amino acid sequences deduced for these three peptides from the nucleotide sequences of the genes have been confirmed, in part, by direct sequencing of portions of the three peptides separated from a sample of the purified, detergent-solubilized complex. These sequences show considerable homology with those previously obtained for the pet operons of other photosynthetic bacteria. Northern blots of R. rubrum mRNA have established that the operon is transcribed as a single polycistronic message, the start site of which has been determined by both primer extension and nuclease protection. Photosynthetic growth of R. rubrum was shown to be inhibited by antimycin A, a specific inhibitor of cytochrome bc 1 complexes, and antimycin A-resistant mutants of R. rubrum have been isolated. Preliminary results suggest that it may be possible to express the R. rubrum pet operon in a strain of the photosynthetic bacterium Rhodobacter capsulatus from which the native pet operon has been deleted.

Mutations conferring resistance to quinol oxidation (Qz) inhibitors of the cyt bc1 complex of Rhodobacter capsulatus.

Several spontaneous mutants of the photosynthetic bacterium Rhodobacter capsulatus resistant to myxothiazol, stigmatellin and mucidin--inhibitors of the ubiquinol: cytochrome c oxidoreductase (cyt bc1 complex)--were isolated. They were grouped into eight different classes based on their genetic location, growth properties and inhibitor cross-resistance. The petABC (fbcFBC) cluster that encodes the structural genes for the Rieske FeS protein, cyt b and cyt c1 subunits of the cyt bc1 complex was cloned out of the representative isolates and the molecular basis of inhibitor-resistance was determined by DNA sequencing. These data indicated that while one group of mutations was located outside the petABC(fbcFBC) cluster, the remainder were single base pair changes in codons corresponding to phylogenetically conserved amino acid residues of cyt b. Of these substitutions, F144S conferred resistance to myxothiazol, T163A and V333A to stigmatellin, L106P and G152S to myxothiazol + mucidin and M140I and F144L to myxothiazol + stigmatellin. In addition, a mutation (aer126) which specifically impairs the quinol oxidase (Qz) activity of the cyt bc1 complex of a non-photosynthetic mutant (R126) was identified to be a glycine to an aspartic acid replacement at position 158 of cyt b. Six of these mutations were found between amino acid residues 140 and 163, in a region linking the putative third and fourth transmembrane helices of cyt b. The non-random clustering of several inhibitor-resistance mutations around the non-functional aer126 mutation suggests that this region may be involved in the formation of the Qz inhibitor binding/quinol oxidation domain(s) of the cyt bc1 complex. Of the two remaining mutations, the V333A replacement conferred resistance to stigmatellin exclusively and was located in another region toward the C terminus of cyt b. The L106P substitution, on the other hand, was situated in the transmembrane helix II that carries two conserved histidine residues (positions 97 and 111 in R. capsulatus) considered to be the axial ligands for the heme groups of cyt b. The structural and functional roles of the amino acid residues involved in the acquisition of Qz inhibitor resistance are discussed in terms of the primary structure of cyt b and in relation to the natural inhibitor-resistance of various phylogenetically related cyt bc/bf complexes.

Improved ultrastructural detail in tissues fixed with potassium permanganate.

An improved method has been developed for fixation with potassium permanganate. Although this is one of the methods widely used to preserve the dense cores of adrenergic storage vesicles, fixation of other tissue components is usually poor. The main differences from previously reported methods using potassium permanganate are the use of a physiological saline as the vehicle for all solutions, and, following this, very rapid dehydration before infiltration with plastic. Cellular and intercellular details of tissue ultrastructure may, in general, be evaluated as satisfactorily as with conventional fixatives, with the exception of certain protein elements associated with ribosome, microtubule, and myofilament organization. Nerve endings with agranular or clear vesicles may be distinguished from adrenergic endings since the dense cores of the vesicles of the latter are preserved by this method.

Regulation of de novo purine biosynthesis in normal and 8-azaguanine-resistant Chinese hamster cells.

Azaguanine-resistant mutants of Chinese hamster ovary cells were isolated following mutagenesis with ICR-17OG. Of the eight mutant isolates examined, only one, Ag-5 had detectable hypoxanthine(guanine)phosphoribosyltransferase activity. Under normal conditions of growth, de novo purine biosynthesis in the mutants was not significantly different from wild type. However, when the cultures were starved for glutamine over a 2 h period before measuring 5'phosphoribosyl formylglycinamide (a relative measure of de novo purine biosynthesis), elevated levels of 5'-phosphoribosyl formylglycinamide accumulated in some of the mutants, and decreased levels in wild type and Ag-5. The level of purine biosynthesis could be related to the levels of glutamine in the pregrowth medium. The rate of purine biosynthesis correlated with 5-phosphoribosyl pyrophosphate levels, which were enhanced in the mutant (Ag-C) following the starvation period. No alterations were found in levels of 5-phosphoribosyl pyrophosphate synthetase or glutamine synthetase. The extent of feedback inhibition was normal in both mutant and wild type cells. These data suggest that the hypoxanthine (guanine) phosphoribosyltransferase locus is a regulatory gene.

Purine mutants of mammalian cell lines: III. Control of purine biosynthesis in adenine phosphoribosyl transferase mutants of CHO cells.

Spontaneous and mutagen-induced 2,6-diaminopurine-resistant mutants of Chinese hamster ovary (CHO-K1) cells were isolated. Such mutants fell into two classes: spontaneous and ethylmethane-sulfonate-induced mutants had approximately 5% wild-type adenine phosphoribosyl transferase (APRT) activity, whereas ICR-170G-induced mutants had barely detectable APRT activity. Since it has been reported that human hypoxanthine-guanine phosphoribosyl transferase (HGPRT) (Lesch-Nyhan syndrome) and APRT mutants over-produce purines, we examined the control and rate of purine biosynthesis in the Chinese hamster mutants. End product inhibition by adenine could not be demonstrated in such mutants, indicating that the active feedback inhibitor is a nucleotide rather than the free purine base, HGPRT activity was normal in all mutants examined except in one isolate. Purine biosynthesis as measured by the accumulation of the purine biosynthetic intermediate phosphoribosyl formylglycineamide was not elevated in the mutants as might have been predicted from work with Lesch-Nyhan cells. The data also suggest that our strain of CHO-K1 is physically or functionally haploid for the APRT locus.