Disruption of Four Kinesin Genes in <it>Dictyostelium</it>

Abstract Background Kinesin and dynein are the two families of microtubule-based motors that drive much of the intracellular movements in eukaryotic cells. Using a gene knockout strategy, we address here the individual function(s) of four of the 13 kinesin proteins in Dictyostelium. The goal of our ongoing project is to establish a minimal motility proteome for this basal eukaryote, enabling us to contrast motor functions here with the often far more elaborate motor families in the metazoans. Results We performed individual disruptions of the kinesin genes, kif4, kif8, kif10, and kif11. None of the motors encoded by these genes are essential for development or viability of Dictyostelium. Removal of Kif4 (kinesin-7; CENP-E family) significantly impairs the rate of cell growth and, when combined with a previously characterized dynein inhibition, results in dramatic defects in mitotic spindle assembly. Kif8 (kinesin-4; chromokinesin family) and Kif10 (kinesin-8; Kip3 family) appear to cooperate with dynein to organize the interphase radial microtubule array. Conclusion The results reported here extend the number of kinesin gene disruptions in Dictyostelium, to now total 10, among the 13 isoforms. None of these motors, individually, are required for short-term viability. In contrast, homologs of at least six of the 10 kinesins are considered essential in humans. Our work underscores the functional redundancy of motor isoforms in basal organisms while highlighting motor specificity in more complex metazoans. Since motor disruption in Dictyostelium can readily be combined with other motility insults and stresses, this organism offers an excellent system to investigate functional interactions among the kinesin motor family.</p

Electronic Structure of Neighboring Extein Residue Modulates Intein C-Terminal Cleavage Activity

Protein splicing is an autocatalytic reaction where an intervening element (intein) is excised and the remaining two flanking sequences (exteins) are joined. The reaction requires specific conserved residues, and activity may be affected by both the intein and the extein sequence. Predicting how sequence will affect activity is a challenging task. Based on first-principles density functional theory and multiscale quantum mechanics/molecular mechanics, we report C-terminal cleavage reaction rates for five mutations at the first residue of the C-extein (+1), and describe molecular properties that may be used as predictors for future mutations. Independently, we report on experimental characterization of the same set of mutations at the +1 residue resulting in a wide range of C-terminal cleavage activities. With some exceptions, there is general agreement between computational rates and experimental cleavage, giving molecular insight into previous claims that the +1 extein residue affects intein catalysis. These data suggest utilization of attenuating +1 mutants for intein-mediated protein manipulations because they facilitate precursor accumulation in vivo for standard purification schemes. A more detailed analysis of the “+1 effect” will also help to predict sequence-defined effects on insertion points of the intein into proteins of interest