MAP1 structural organization in Drosophila: in vivo analysis of FUTSCH reveals heavy- and light-chain subunits generated by proteolytic processing at a conserved cleavage site

The MAP1 (microtubule-associated protein 1) family is a class of microtubule-binding proteins represented by mammalian MAP1A, MAP1B and the recently identified MAP1S. MAP1A and MAP1B are expressed in the nervous system and thought to mediate interactions of the microtubule-based cytoskeleton in neural development and function. The characteristic structural organization of mammalian MAP1s, which are composed of heavy- and light-chain subunits, requires proteolytic cleavage of a precursor polypeptide encoded by the corresponding map1 gene. MAP1 function in Drosophila appears to be fulfilled by a single gene, futsch. Although the futsch gene product is known to share several important functional properties with mammalian MAP1s, whether it adopts the same basic structural organization has not been addressed. Here, we report the identification of a Drosophila MAP1 light chain, LC(f), produced by proteolytic cleavage of a futsch-encoded precursor polypeptide, and confirm co-localization and co-assembly of the heavy chain and LC(f) cleavage products. Furthermore, the in vivo properties of MAP1 proteins were further defined through precise MS identification of a conserved proteolytic cleavage site within the futsch-encoded MAP1 precursor and demonstration of light-chain diversity represented by multiple LC(f) variants. Taken together, these findings establish conservation of proteolytic processing and structural organization among mammalian and Drosophila MAP1 proteins and are expected to enhance genetic analysis of conserved MAP1 functions within the neuronal cytoskeleton.     

Heterotypic complex formation between subunits of microtubule-associated proteins 1A and 1B is due to interaction of conserved domains

The microtubule-associated proteins MAP1A and MAP1B are related but distinct multi-subunit protein complexes that consist of heavy and light chains. The predominant forms of these complexes are homotypic, i.e. they consist of a MAP1A heavy chain associated with MAP1A light chains or a MAP1B heavy chain associated with MAP1B light chains, respectively. In addition, MAP1A and MAP1B can exchange subunits and form heterotypic complexes consisting of a MAP1A heavy chain associated with MAP1B light chains which might play a role in a transition period of neuronal differentiation. Here we extend previous findings by confirming that heterotypic MAP1B heavy chain-MAP1A light chain complexes also exist in the developing murine brain. We show that these complexes form through interaction of homologous domains conserved in heavy and light chains of MAP1A and MAP1B. Likewise, conserved domains of the MAP1A and MAP1B light chains account for formation of light chain heterodimers. By yeast 2-hybrid analysis we located the light chain binding domain on the heavy chain to amino acids 211-508, thereby defining a new functional subdomain.     

Novel Features of the Light Chain of Microtubule-associated Protein MAP1B: Microtubule Stabilization,Self Interaction,Actin Filament Binding,and Regulation by the Heavy Chain

Previous studies on the role of microtubule-associated protein 1B (MAP1B) in adapting microtubules for nerve cell-specific functions have examined the activity of the entire MAP1B protein complex consisting of heavy and light chains and revealed moderate effects on microtubule stability. Here we have analyzed the effects of the MAP1B light chain in the absence or presence of the heavy chain by immunofluorescence microscopy of transiently transfected cells. Distinct from all other MAPs, the MAP1B light chain–induced formation of stable but apparently flexible microtubules resistant to the effects of nocodazole and taxol. Light chain activity was inhibited by the heavy chain. In addition, the light chain was found to harbor an actin filament binding domain in its COOH terminus. By coimmunoprecipitation experiments using epitope-tagged fragments of MAP1B we showed that light chains can dimerize or oligomerize. Furthermore, we localized the domains for heavy chain–light chain interaction to regions containing sequences homologous to MAP1A. Our findings assign several crucial activities to the MAP1B light chain and suggest a new model for the mechanism of action of MAP1B in which the heavy chain might act as the regulatory subunit of the MAP1B complex to control light chain activity.     

Microtubule-associated protein 1S, a short and ubiquitously expressed member of the microtubule-associated protein 1 family

The related high molecular mass microtubule-associated proteins (MAPs) MAP1A and MAP1B are predominantly expressed in the nervous system and are involved in axon guidance and synaptic function. MAP1B is implicated in fragile X mental retardation, giant axonal neuropathy, and ataxia type 1. We report the functional characterization of a novel member of the microtubule-associated protein 1 family, which we termed MAP1S (corresponding to sequence data bank entries for VCY2IP1 and C19ORF5). MAP1S contains the three hallmark domains of the microtubule-associated protein 1 family but hardly any additional sequences. It decorates neuronal microtubules and copurifies with tubulin from brain. MAP1S is synthesized as a precursor protein that is partially cleaved into heavy and light chains in a tissue-specific manner. Heavy and light chains interact to form the MAP1S complex. The light chain binds, bundles, and stabilizes microtubules and binds to actin. The heavy chain appears to regulate light chain activity. In contrast to MAP1A and MAP1B, MAP1S is expressed in a wide range of tissues in addition to neurons and represents the non-neuronal counterpart of this cytolinker family.     

A model for microtubule-associated protein 4 structure. Domains defined by comparisons of human, mouse, and bovine sequences.

cDNAs encoding human and mouse microtubule-associated protein 4 (MAP 4) were isolated. MAP 4 is encoded by a single gene. Multiple MAP 4 mRNAs are transcribed that are differentially expressed among mouse tissues. Open reading frames for the human and mouse MAP 4 clones indicate three distinct regions consisting of related sequences with different motifs. Approximately 30% of the protein is tandem related repeats of approximately 14 amino acids. Another region contains clusters of serine and proline. Four 18-mer repeats characteristic of the microtubule-binding domains of MAP 2 and tau are located at the carboxyl-terminal portion of MAP 4. Amino acid sequence analysis revealed that human and mouse MAP 4 are homologs of the bovine 190-kDa MAP/MAP U (Aizawa, H., Emori, Y., Murofushi, H., Kawasakai, H., Sakai, H., and Suzuki, K. (1990) J. Biol. Chem. 265, 13849-13855). Mouse and human MAP 4 and the bovine 190-kDa MAP are approximately 75% similar, indicating that these proteins are all members of the same class. Domains with extremely high conservation (greater than or equal to 88%) are: 1) the extreme amino terminus; 2) a proline-rich region between the KDM and S,P domains; 3) the microtubule-binding domain; and 4) the extreme carboxyl terminus.     

The MAP1 family of microtubule-associated proteins

MAP1-family proteins are classical microtubule-associated proteins (MAPs) that bind along the microtubule lattice. The founding members, MAP1A and MAP1B, are predominantly expressed in neurons, where they are thought to be important in the formation and development of axons and dendrites. Mammalian genomes usually contain three family members, MAP1A, MAP1B and a shorter, more recently identified gene called MAP1S. By contrast, only one family member, Futsch, is found in Drosophila. After their initial expression, the MAP1A and MAP1B polypeptides are cleaved into light and heavy chains, which are then assembled into mature complexes together with the separately encoded light chain 3 subunit (LC3). Both MAP1A and MAP1B are well known for their microtubule-stabilizing activity, but MAP1 proteins can also interact with other cellular components, including filamentous actin and signaling proteins. Furthermore, the activity of MAP1A and MAP1B is controlled by upstream signaling mechanisms, including the MAP kinase and glycogen synthase kinase-3 β pathways.     

MAP 1C is a microtubule-activated ATPase which translocates microtubules in vitro and has dynein-like properties

We observe that one of the high molecular mass microtubule-associated proteins (MAPs) from brain exhibits nucleotide-dependent binding to microtubules. We identify the protein as MAP IC, which was previously described in this laboratory as a minor component of standard microtubule preparations (Bloom, G.S., T. Schoenfeld, and R.B. Vallee, 1984, J. Cell Biol., 98:320-330). We find that MAP 1C is enriched in microtubules prepared in the absence of nucleotide. Kinesin is also found in these preparations, but can be specifically extracted with GTP. A fraction highly enriched in MAP 1C can be prepared by subsequent extraction of the microtubules with ATP. Two activities cofractionate with MAP 1C upon further purification, a microtubule-activated ATPase activity and a microtubule-translocating activity. These activities indicate a role for the protein in cytoplasmic motility. MAP 1C coelectrophoreses with the beta heavy chain of Chlamydomonas flagellar dynein, and has a sedimentation coefficient of 20S. Exposure to ultraviolet light in the presence of vanadate and ATP results in the production of two large fragments of MAP 1C. These characteristics suggest that MAP 1C may be a cytoplasmic analogue of axonemal dynein.     

A plant kinesin heavy chain-like protein is a calmodulin-binding protein

Calmodulin, a calcium modulated protein, regulates the activity of several proteins that control cellular functions. A cDNA encoding a unique calmodulin-binding protein, PKCBP, was isolated from a potato expression library using protein-protein interaction based screening. The cDNA encoded protein bound to biotinylated calmodulin and ³⁵S-labeled calmodulin in the presence of calcium and failed to bind in the presence of EGTA, a calcium chelator. The deduced amino acid sequence of the PKCBP has a domain of about 340 amino acids in the C-terminus that showed significant sequence similarity with the kinesin heavy chain motor domain and contained conserved ATP- and microtubule-binding sites present in the motor domain of all known kinesin heavy chains. Outside the motor domain, the PKCBP showed no sequence similarity with any of the known kinesins, but contained a globular domain in the N-terminus and a putative coiled-coil region in the middle. The calmodulin-binding region was mapped to a stretch of 64 amino acid residues in the C-terminus region of the protein. The gene is differentially expressed with the highest expression in apical buds. A homolog of PKCBP from Arabidopsis (AKCBP) showed identical structural organization indicating that kinesin heavy chains that bind to calmodulin are likely to exist in other plants. This paper presents evidence that the motor domain has microtubule stimulated ATPase activity and binds to microtubules in a nucleotide-dependent manner. The kinesin heavy chain-like calmodulin-binding protein is a new member of the kinesin superfamily as none of the known kinesin heavy chains contain a calmodulin-binding domain. The presence of a calmodulin-binding motif and a motor domain in a single polypeptide suggests regulation of kinesin heavy chain driven motor function(s) by calcium and calmodulin.     

The functional cooperation of MAP1A heavy chain and light chain 2 in the binding of microtubules

Microtubule-associated protein 1A (MAP1A) is a high-molecular-weight protein that is comprised of a heavy chain and a light chain (LC2) and is widely distributed along the microtubules in both mature neurons and glial cells. To illustrate the interaction among the MAP1A heavy chain, light chain, and microtubule, we prepared DNA constructs with Myc-, EGFP-, or DsRed-tags for full-length MAP1A DNA expressing whole MAP1A protein, two domains of MAP1A heavy chain, and light chain. Distribution patterns of various MAP1A domains as well as their interactions with microtubules were monitored in a non-neuronal COS7 and a neuronal Neuro2A cells. Our data revealed that a complete MAP1A protein, which contains both heavy chain and LC2, could be colocalized with microtubule networks not only in Neuro2A cells but also in transfected COS7 cells. Filamentous structures failed to be visualized along microtubules in COS7 cells transfected with MAP1A heavy chain or LC2 alone. Whereas, after introducing MAP1A heavy chain with LC2 into COS7 cells, both heavy chain and LC2 could be colocalized with microtubules. From our functional analysis, both MAP1A and its LC2 could protect microtubules against the challenge of nacodazol. Data collected from yeast two-hybrid assays of various MAP1A domains confirmed that the interaction of LC2 and NH2-terminal of MAP1A heavy chain is important for microtubule binding. From our analysis of MAP1A functional domains, we suggest that interactions between MAP1A heavy chain and LC2 are critical for the binding of microtubules.     

A split motor domain in a cytoplasmic dynein

The heavy chain of dynein forms a globular motor domain that tightly couples the ATP-cleavage region and the microtubule-binding site to transform chemical energy into motion along the cytoskeleton. Here we show that, in the fungus Ustilago maydis, two genes, dyn1 and dyn2, encode the dynein heavy chain. The putative ATPase region is provided by dyn1, while dyn2 includes the predicted microtubule-binding site. Both genes are located on different chromosomes, are transcribed into independent mRNAs and are translated into separate polypeptides. Both Dyn1 and Dyn2 co-immunoprecipitated and co-localized within growing cells, and Dyn1-Dyn2 fusion proteins partially rescued mutant phenotypes, suggesting that both polypeptides interact to form a complex. In cell extracts the Dyn1-Dyn2 complex dissociated, and microtubule affinity purification indicated that Dyn1 or associated polypeptides bind microtubules independently of Dyn2. Both Dyn1 and Dyn2 were essential for cell survival, and conditional mutants revealed a common role in nuclear migration, cell morphogenesis and microtubule organization, indicating that the Dyn1-Dyn2 complex serves multiple cellular functions.     

Silk fibroin of Bombyx mori is secreted, assembling a high molecular mass elementary unit consisting of H-chain, L-chain, and P25, with a 6:6:1 molar ratio

Silk fibroin produced by the silkworm Bombyx mori consists of a heavy chain, a light chain, and a glycoprotein, P25. The heavy and light chains are linked by a disulfide bond, and P25 associates with disulfide-linked heavy and light chains by noncovalent interactions. Quantitative enzyme-linked immunosorbent assay revealed that molar ratios of the heavy chain, light chain, and P25 were 6:6:1, both in cocoons and in fibroin secreted into the lumen of posterior silk gland. Trace amounts of fibroin produced by three "naked pupa" mutants of B. mori lacked the light chain, but the molar ratio of heavy chain and P25 was also 6:1. Gel filtration chromatography and sedimentation equilibrium analysis demonstrated that a large protein complex of approximately 2.3 MDa, designated an elementary unit of fibroin having 6:6:1 molar ratios of the heavy chain, light chain, and P25, existed in posterior silk gland cells. Inaccessibility of biotinylated concanavalin A to the native elementary unit and partial dissociation of the elementary unit after incubation with excess N-glycosidase F or endoglycosidase H suggest that a single molecule of P25 is located internally and plays an important role in maintaining integrity of the complex.     

Thrombin cleavage analysis of a novel antihaemophilic factor variant, factor VIII delta II

Factor VIII delta II is a genetically engineered deletion variant of factor VIII expressed by recombinant Chinese hamster ovary cells, in which a major portion of the central (B) domain and a part of the light chain (Pro771-Asp1666) are missing. After immunoaffinity purification, the kinetics of thrombin cleavage of the novel molecule was analysed by SDS/PAGE, Western blotting and N-terminal amino acid sequencing. Thrombin first cleaves factor VIII delta II at Arg740-Ser741 to generate the 90-kDa heavy chain and an 80-kDa fusion polypeptide consisting of the remaining portion of the B domain and the 73-kDa light chain. The 90-kDa fragment is further cleaved, giving rise to 50-kDa and 40-kDa fragments while the 80-kDa fragment generates a 71/73-kDa doublet. The 71/73-kDa doublet, 50-kDa and 40-kDa fragments were further analysed by N-terminal amino acid sequencing and found to correspond to the predicted amino acid sequences. Our study shows that, in spite of the 900 amino acid deletion present in factor VIII delta II, the essential structural elements required for thrombin activation are conserved.     

Identification of a neural cell specific variant of microtubule-associated protein 4

The microtubule-binding domain of MAP4, a ubiquitous microtubule-associated protein, contains a region rich in proline and basic residues (proline-rich region). We searched the bovine adrenal gland for MAP4 isoforms, and identified a novel variant lacking 72 consecutive amino acid residues within the proline-rich region, as compared with the full-length MAP4. The amino acid sequence of the missing region was highly conserved (about 85% identity/similarity) among the corresponding regions of bovine, human, mouse, and rat MAP4, which suggested the functional significance of this region. A comparison of the genomic sequence with the cDNA sequence revealed that the missing region is encoded by a single exon. A MAP4 variant cDNA homologous to the bovine form was also detected in rat cells, suggesting that the new variant can be generated by alternative splicing, not only in bovine but also in other mammalian species. The mRNA expression of the novel isoform was restricted to the brain and the adrenal medulla, suggesting that this isoform is specific to a certain cell type. Using a bacterially expressed fragment corresponding to the microtubule-binding domain of the novel isoform, we analyzed its in vitro characteristics. The fragment induced microtubule assembly and bound to preformed microtubules, but the activities were slightly lower than those of the conventional MAP4 fragment, which carries the full-length proline-rich region. The microtubules assembled in the presence of the fragment failed to be bundled. Instead, a constant spacing between neighboring microtubules was observed.     

Calcium regulates ATP-sensitive microtubule binding by Chlamydomonas outer arm dynein

The Chlamydomonas outer dynein arm contains three distinct heavy chains (alpha, beta, and gamma) that exhibit different motor properties. The LC4 protein, which binds 1-2 Ca2+ with KCa = 3 x 10-5 m, is associated with the gamma heavy chain and has been proposed to act as a sensor to regulate dynein motor function in response to alterations in intraflagellar Ca2+ levels. Here we genetically dissect the outer arm to yield subparticles containing different motor unit combinations and assess the microtubule-binding properties of these complexes both prior to and following preincubation with tubulin and ATP, which was used to inhibit ATP-insensitive (structural) microtubule binding. We observed that the alpha heavy chain exhibits a dominant Ca2+-independent ATP-sensitive MT binding activity in vitro that is inhibited by attachment of tubulin to the structural microtubule-binding domain. Furthermore, we show that ATP-sensitive microtubule binding by a dynein subparticle containing only the beta and gamma heavy chains does not occur at Ca2+ concentrations below pCa 6 but is maximally activated above pCa 5. This activity was not observed in mutant dyneins containing small deletions in the microtubule-binding region of the beta heavy chain or in dyneins that lack both the alpha heavy chain and the motor domain of the beta heavy chain. These findings strongly suggest that Ca2+ binding directly to a component of the dynein complex regulates ATP-sensitive interactions between the beta heavy chain and microtubules and lead to a model for how individual motor units are controlled within the outer dynein arm.     

Clathrin promotes centrosome integrity in early mitosis through stabilization of centrosomal ch-TOG

Clathrin depletion by ribonucleic acid interference (RNAi) impairs mitotic spindle stability and cytokinesis. Depletion of several clathrin-associated proteins affects centrosome integrity, suggesting a further cell cycle function for clathrin. In this paper, we report that RNAi depletion of CHC17 (clathrin heavy chain 17) clathrin, but not the CHC22 clathrin isoform, induced centrosome amplification and multipolar spindles. To stage clathrin function within the cell cycle, a cell line expressing SNAP-tagged clathrin light chains was generated. Acute clathrin inactivation by chemical dimerization of the SNAP-tag during S phase caused reduction of both clathrin and ch-TOG (colonic, hepatic tumor overexpressed gene) at metaphase centrosomes, which became fragmented. This was phenocopied by treatment with Aurora A kinase inhibitor, suggesting a centrosomal role for the Aurora A-dependent complex of clathrin, ch-TOG, and TACC3 (transforming acidic coiled-coil protein 3). Clathrin inactivation in S phase also reduced total cellular levels of ch-TOG by metaphase. Live-cell imaging showed dynamic clathrin recruitment during centrosome maturation. Therefore, we propose that clathrin promotes centrosome maturation by stabilizing the microtubule-binding protein ch-TOG, defining a novel role for the clathrin-ch-TOG-TACC3 complex.     

GRIM-19, a cell death regulatory gene product, is a subunit of bovine mitochondrial NADH:ubiquinone oxidoreductase (complex I)

The sequences of 42 subunits of NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria have been described previously. Seven are encoded by mitochondrial DNA, whereas the remaining 35 are nuclear gene products imported into the organelle from the cytoplasm. An additional protein, which does not correspond to any previously known subunit of the complex I assembly, has now been detected. Denaturing gels of subcomplex Ilambda, the hydrophilic arm of complex I, clearly show a hitherto unidentified band, which was digested with trypsin and subjected to mass-spectrometric analysis to provide several peptide sequences, used in cDNA cloning and sequencing. Measurement of the intact protein mass indicated that the N terminus is acetylated. The new complex I subunit (B16.6) is the bovine homolog of GRIM-19, the product of a cell death regulatory gene induced by interferon-beta and retinoic acid, thus providing a new link between the mitochondrion and its electron-transport chain and apoptotic cell death.     

Two kinesins from Arabidopsis, KatB and KatC, have a second microtubule-binding site in the tail domain

Kinesins, as a kind of microtubule-based motor proteins, have a conserved microtubule-binding site in their motor domain. Here we report that two homologous kinesins in Arabidopsis thaliana, KatB and KatC, contain a second microtubule-binding site in their tail domains. The prokaryotic-expressed N-terminal tail domain of the KatC heavy chain can bind to microtubules in an ATP-insensitive manner. To identify the precise region responsible for the binding, a serious of truncated KatC cDNAs encoding KatC N-terminal regions in different lengths, KatC1-128, KatC1-86, KatC1-73 and KatC1-63, fused to Histidine-tags, were expressed in E. coli and affinity-purified. Microtubule cosedimentation assays show that the site at amino acid residues 74-86 in KatC is important for microtubulebinding. By similarity, we obtained three different lengths of KatB N-terminal regions, KatB1-384, KatB1-77, and KatB1-63, and analyzed their microtubule-binding ability. Cosedimentation assays indicate that the KatB tail domain can also bind to microtubules at the same site as and in a similar manner to KatC. Fluorescence microscopic observations show that the microtubule-binding site at the tail domain of KatB or KatC can induce microtubules bundling only when the stalk domain is present. Through pull-down assays, we show that KatB1-385 and KatC1-394 are able to interact specifically with themselves and with each other in vitro. These findings are significant for identifying a previously uncharacterized microtubule-binding site in the two kinesin proteins, KatB and KatC, and the functional relations between them.     

DNA sequence of Mirabilis antiviral protein (MAP), a ribosome-inactivating protein with an antiviral property, from mirabilis jalapa L. and its expression in Escherichia coli

We cloned a cDNA for Mirabilis antiviral protein (MAP), a ribosome-inactivating protein (RIP), which inhibits the mechanical transmission of plant virus and the in vitro protein synthesis of both prokaryotes and eukaryotes. The cDNA consisted of 1066 nucleotides and could encode 278 amino acids. The major part of the amino acid sequence (from Ala29 to Ser278) was identical with the sequence of native MAP as determined by protein sequencing. An NH2-terminal extrapeptide (28 amino acid residues) of MAP was comparable with the signal peptides of plant proteins accumulating in the vacuole. A stable hairpin structure was predicted in the 3'-noncoding region of the cDNA. Tandem repeated sequences were found downstream from the hairpin structure. They were composed of triple complete repeats of a heptanucleotide with preceding and following hexa-nucleotide repeats. The cDNA was expressed in Escherichia coli based on the T7 expression system. The product encoded by the cDNA was confirmed to be MAP precursor by Western blotting followed by immunological analysis. The growth of the transformants was inhibited by the expression of the gene. MAP precursor also seemed to inhibit the protein synthesis of E. coli just as native MAP has been observed to do.