Journal of Cell Science 113, 1311-1318 (2000) 1311 Printed in Great Britain © The Company of Biologists Limited 2000 JCS0832 COMMENTARY Molecular motors − a paradigm for mutant analysis Sharyn A. Endow Department of Microbiology, Duke University Medical Center, Durham, NC 27710 USA (e-mail: [email protected]) Published on WWW 21 March 2000 SUMMARY Molecular motors perform essential functions in the cell or by rational design. Mutants that are expected to and have the potential to provide insights into the basis provide important information about the motor of many important processes. A unique property of mechanism include ATPase mutants, which interfere with molecular motors is their ability to convert energy from the nucleotide hydrolysis cycle, and uncoupling mutants, ATP hydrolysis into work, enabling the motors to bind to which unlink basic motor activities and reveal their and move along cytoskeletal filaments. The mechanism interdependence. Natural variants can also be exploited of energy conversion by molecular motors is not yet to provide unexpected information about motor function. understood and may lead to the discovery of new This general approach to uncovering protein function by biophysical principles. Mutant analysis could provide analysis of informative mutants is applicable not only to valuable information, but it is not obvious how to obtain molecular motors, but to other proteins of interest. mutants that are informative for study. The analysis presented here points out several strategies for obtaining mutants by selection from molecular or genetic screens, Key words: Molecular motor, Mutant, Structure/function INTRODUCTION The cellular roles of the motors and interactions between the motors are only beginning to be understood. The fact that Molecular motors carry out a myriad of functions in the cell, the cellular functions of many of the motors are as yet ranging from vesicle (Hirokawa, 1998) and RNA transport unknown means that the effects of mutants that cause loss of (Nasmyth and Jansen, 1997) to spindle assembly and function have not always been straightforward to predict, and chromosome motility in dividing cells (Endow, 1999c). The some of these effects have been unexpected. For example, finding of a large number of motors that fall into one of the mutants of the unconventional myosins, myosin VI and VII, three families of motors known to exist in eukaryotes, the actin- cause deafness in mice and humans, and are the basis of based myosins (Sellers and Goodson, 1995) or microtubule- previously described syndromes, Snell’s Walzer and shaker, based kinesins (Bloom and Endow, 1995) and dyneins that are associated with behavioral abnormalities in mice (Hirokawa, 1998), has been revolutionary in permitting (Avraham et al., 1995; Gibson et al., 1995; Weil et al., 1995). analysis of diverse biological processes as the roles of the These myosins are present in hair cells of the inner ear, where proteins in the cell are discovered. The importance of their roles in hearing are currently being investigated. molecular motors in the cell is exemplified by the process of Another example of an unexpected effect of a mutant comes cell division, in which many of the kinesin microtubule motors, from work on the kinesin-related KIF3B microtubule motor. together with cytoplasmic dynein, produce force for spindle In the KIF3B-knockout mouse, loss of KIF3B is associated assembly and chromosome attachment to the spindle (Endow, with loss of bilateral asymmetry (Nonaka et al., 1998), which 1999c). Cytoplasmic myosin then functions during cytokinesis is needed to form organs such as the heart in mammals. to complete the process of cell division by accumulating in the Hirokawa and co-workers propose that the basis of the loss cleavage furrow and providing motile force for the contractile of asymmetry is defective transport of substances required for ring (Goldberg et al., 1998). Vesicle trafficking similarly formation of cilia in the early embryo (Nonaka et al., 1998). involves motors from all three families (Hirokawa, 1998; They hypothesize that the cilia cause a leftward flow of Brown, 1999), while specific cellular roles are played by extraembryonic fluid in the area of the node, which is a myosin in muscle contraction (Cooke, 1986), dyneins in transient structure present during gastrulation, and therefore flagellar motility (Gibbons, 1996) and kinesins in regulation of play a key role in the formation of left-right morphogen microtubule dynamics (Inoué and Salmon, 1995; Waters and gradients. These unforeseen mutant effects reflect the Salmon, 1996). inability to predict the cellular roles of the newly discovered 1312 S. A. Endow myosin, kinesin and dynein motors, and underscore the different from other nucleotide-hydrolyzing proteins in necessity for cellular studies. But to fully understand the roles possessing the ability to convert chemical energy into work has of molecular motors in cells, it is necessary to determine how led to the question of how to obtain information about the motor the motors work. mechanism. Strategies that have been used successfully for other proteins, such as the G proteins, are to characterize the proteins functionally and structurally using available biochemical and THE MOTOR MECHANISM structural methods (Bourne et al., 1991), and then to mutate specific residues of the proteins and determine the effects of the Molecular motors are unique in their ability to convert the mutations on protein structure and function (Raw et al., 1997). chemical energy from ATP hydrolysis into work, which permits Findings from such structure/function studies have resulted in them to bind to and move along actin filaments or microtubules. working models of G protein function (Sprang, 1997). Further The process of energy conversion by motor proteins is not tests by state-of-the-art methods, such as time-resolved or understood, and considerable effort is now being devoted to millisecond-resolution X-ray crystallography (Scott et al., 1996; unraveling the possible mechanisms. The discovery of large Stoddard et al., 1998), could confirm the conformational changes families of related motors whose members differ from one in protein structure proposed to occur during the hydrolysis cycle, another in directionality of movement on their filament, ATPase which are based on the ‘still’ images from static crystal structures. activity and motor velocity offers an unprecedented opportunity This general approach is one that is being used to study the to perform studies to uncover the mechanism by which these mechanism of myosin function and has led to several proteins function. This information will be valuable, first, noteworthy achievements in recent years. The solution of the because it represents basic information about energy conversion first myosin motor domain structure was a breakthrough in living organisms and may lead to the discovery of new (Rayment et al., 1993) that has been rapidly followed by new principles and, second, because it will help us to understand myosin structures (Fisher et al., 1995; Dominguez et al., 1998; how the motors work in the cell. Practical uses of the anticipated Houdusse et al., 1999), as noted above. Mutant analysis coupled information will be to design mutants that disrupt specific with expression of functional myosin proteins in baculovirus is cellular functions, or to create motors for transport of drugs or underway (Ruppel and Spudich, 1995) and should provide other substances to specific cells or regions of cells. information about the myosin mechanism of function. But A milestone towards the goal of understanding the changes despite the recent surge in progress on myosin, the kinesins that take place in the motors during the conversion of chemical have continued to attract attention. The kinesins offer several to mechanical energy has been the solution of crystal structures advantages over myosin, including the smaller size of the of myosin in three different nucleotide states: a ‘rigor’ state motors compared with myosin, which is a distinct advantage for without nucleotide (Rayment et al., 1993), a putative transition structural analysis, and the ease of expressing the kinesin − state with bound ADP⋅AlF4 (Fisher et al., 1995; Dominguez et proteins in bacteria compared with the technicalities of al., 1998), and a third distinct conformational state that may baculovirus expression. On the other hand, a major barrier to correspond to a prehydrolysis state (Houdusse et al., 1999). A rapid progress on the kinesins has been the inability to comparable advance for the kinesins has been the solution of crystallize the motors in different conformations that crystal structures of monomeric and dimeric forms of the motors correspond to different nucleotide states of the motor. Although in the Mg⋅ADP state (Sack et al., 1999). The crystal structures seven crystal structures of kinesin proteins have been reported of the kinesins provide information about the dimeric forms of to date (Sack et al., 1999; Kozielski et al., 1999), all of the the motors, currently unavailable for the myosins, but are structures are in the same nucleotide state, with bound Mg⋅ADP. uninformative about the changes that take place in the motors Efforts to identify different structures of the kinesins bound to during nucleotide hydrolysis because they all correspond to the other nucleotides, including attempts to use nucleotide same state, presumably a post-hydrolysis state. Nonetheless, the analogues to grow crystals of motors in