Lawrence S.B. Goldstein

A three-domain structure of kinesin heavy chain revealed by DNA sequence and microtubule binding analyses

The structure and function of kinesin heavy chain from D. melanogaster have been studied using DNA sequence analysis and analysis of the properties of truncated kinesin heavy chain synthesized in vitro. Analysis of the sequence suggests the existence of a 50 kd globular amino-terminal domain that contains an ATP binding consensus sequence, followed by another 50-60 kd domain that has sequence characteristics consistent with the ability to fold into an alpha helical coiled coil. The properties of amino- and carboxy-terminally truncated kinesin heavy chains synthesized in vitro reveal that a 60 kd amino-terminal fragment has the nucleotide-dependent microtubule binding activities of the intact kinesin heavy chain, and hence is likely to be a motor domain. Finally, the sequence data indicate the presence of a small carboxy-terminal domain. Because it is located at the end of the molecule away from the putative motor domain, we propose that this domain is responsible for interactions with other proteins, vesicles, or organelles. These data suggest that kinesin has an organization very similar to that of myosin even though there are no obvious sequence similarities between the two molecules.

The kinesin-like ncd protein of Drosophila is a minus end-directed microtubule motor

The Drosophila ncd gene is required for chromosome segregation during female meiosis. Previous analyses suggested that the ncd gene encoded a protein with sequence similarity to the kinesin motor domain, which suggested that, like kinesin, the ncd protein might be a plus end-directed microtubule motor. Here we describe the expression of ncd protein in E. coli and the initial characterization of the ncd proteins motor properties. The ncd protein is indeed a microtubule motor, but the polarity of movement is minus end directed. The ncd protein also has microtubule bundling activity. These findings limit possible models for the in vivo functions of the ncd protein and suggest that motor proteins with similar sequence can generate movement in opposite directions along a microtubule.

A kinesin-like protein required for distributive chromosome segregation in Drosophila

The nod gene is required for the distributive segregation of nonexchange chromosomes during meiosis in D. melanogaster. Loss-of-function nod mutations cause nondisjunction and loss of nonrecombinant chromosomes both at meiosis I and during subsequent mitotic divisions. We have cloned the nod locus, examined its expression patterns, and determined its coding sequence. In adults the nod transcript is only present in females, consistent with the observation that males do not use the distributive segregation system. However, the nod locus is also transcribed in the embryonic, larval, and pupal stages of development, and possibly in all dividing cells. Finally, the N-terminal domain of the predicted nod protein has amino acid similarity to the mechanochemical domain of kinesin heavy chain; however, the C-terminal domain is unlike that of kinesin heavy chain or of any previously reported protein. Thus, the nod protein is a member of the kinesin superfamily and may be a microtubule motor.

Kinesin Heavy Chain Is Essential for Viability and Neuromuscular Functions in Drosophila, but Mutants Show No Defects in Mitosis

The in vivo function of the microtubule motor protein kinesin was examined in Drosophila using genetics and immunolocalization. Kinesin heavy chain mutations (khc) cause abnormal behavior and lethality. Mutant larvae exhibit loss of mobility and tactile responsiveness in the most posterior segments, followed by general paralysis and death during larval or pupal development. Adults homozygous for a temperature-sensitive allele also exhibit a loss in mobility and sensory responses. The data indicate that kinesin function is essential and suggest that kinesin has an important role in the neuromuscular system, perhaps as a motor for axonal transport. The possibility of more general cellular functions remains open, but observation of embryogenesis and morphogenesis in khc mutants suggests that mitosis and the cell cycle can proceed in spite of impaired kinesin function. Immunolocalization suggests that kinesin may have some general cellular functions but that it is not a major component of mitotic spindles.

Identification and characterization of a gene encoding a kinesin-like protein in Drosophila

We identified and sequenced a cDNA clone encoding a kinesin-like protein from Drosophila. The predicted product of this cDNA has a carboxy-terminal domain that is substantially similar to the motor domain of kinesin heavy chain. The amino-terminal domain is unlike that found in previously identified kinesins or kinesin-like proteins. Analyses of this new sequence suggest that the maximal motor unit in the kinesin superfamily may be as little as 350 amino acids, and that the existence of both kinesin and kinesin-like molecules must be an evolutionarily ancient feature of eukaryotes. We also tested some of the biochemical properties of the protein encoded by this cDNA and found them to be similar to those of kinesin. Finally, the clone we isolated appears to correspond to the non-claret disjunctional (ncd) gene, which when mutant causes defects in meiotic and early embryonic mitotic chromosome segregation, and whose recently determined sequence predicts a kinesin-like domain.

The kinesin superfamily: tails of functional redundancy

Kinesin is a microtubule-based motility protein that mediates axonal transport and perhaps other intracellular movements in eukaryotic cells. Recent research has indicated that the principal component of kinesin, the kinesin heavy chain, is but one member of an extended superfamily of kinesin-like microtubule motor proteins. These proteins appear to have diverse microtubule-based motility functions--in mitosis, meiosis, vesicle transport and organelle transport. The various kinesin-like molecules may play overlapping or redundant roles in these processes.

Identification of the gene for fly non-muscle myosin heavy chain: Drosophila myosin heavy chains are encoded by a gene family.

In contrast to vertebrate species Drosophila has a single myosin heavy chain gene that apparently encodes all sarcomeric heavy chain polypeptides. Flies also contain a cytoplasmic myosin heavy chain polypeptide that by immunological and peptide mapping criteria is clearly different from the major thoracic muscle isoform. Here, we identify the gene that encodes this cytoplasmic isoform and demonstrate that it is distinct from the muscle myosin heavy chain gene. Thus, fly myosin heavy chains are the products of a gene family. Our data suggest that the contractile function required to power myosin based movement in non‐muscle cells requires myosin diversity beyond that available in a single heavy chain gene. In addition, we show, that accumulation of cytoplasmic myosin transcripts is regulated in a developmental stage specific fashion, consistent with a key role for this protein in the movements of early embryogenesis.

Chapter 1 Molecular Genetic Analyses of Drosophila Kinesin

Publisher Summary Intracellular movements are necessary for the establishment of cytoplasmic order and also for communication, between the specialized compartments of the cytoplasm. These needs are partly fulfilled by a growing list of cytoskeleton-associated mechanochemical proteins. These mechanochemical proteins hydrolyze adenosine triphosphate (ATP) and move along cytoskeletal filaments, either actin or microtubules. Kinesin is a microtubule-associated mechanochemical protein complex. The occurrence of kinesin in most eukaryotic cell types suggests that kinesin plays a role in the spatial organization of the eukaryotic cytoplasm. Several kinesin-like proteins, with defined intracellular motility functions, have been discovered, using genetic and molecular genetic methods. The discovery of these kinesin-like genes suggests that there probably exists a superfamily of kinesin-like proteins with dozens of members that perform specialized intracellular motility functions. The members of this superfamily are related by possessing a common microtubule-associated mechanochemical domain. All the members of this super-family discovered, thus, have far specialized domains that are likely to associate with specific cellular components. This chapter discusses the kinesin literature relevant to kinesin structure and also discusses the results, concerning the structure, and the in vitro motility properties of genetically manipulated forms of the Drosophila kinesin heavy chain. The chapter describes the biochemical evidence that the kinesin mechanochemical head domain is a functional unit independent of additional domains to which it can be attached. The analysis explained in the chapter, with the finding that there exists a superfamily of proteins with domains that are similar to the N-terminal domain of the kinesin heavy chain, suggests that the kinesin head constitutes a functionally autonomous, microtubule-associated mechanochemical domain that is independent of additional domains to which it is attached and also of its orientation within the linear sequence of the molecule.