A Developmentally Regulated Kinesin-related Motor Protein fromDictyostelium discoideum

Abstract

The cellular slime mold Dictyostelium discoideum is an attractive system for studying the roles of microtubule-based motility in cell development and differentiation. In this work, we report the first molecular characterization of kinesin-related proteins (KRPs) in Dictyostelium. A PCR-based strategy was used to isolate DNA fragments encoding six KRPs, several of which are induced during the developmental program that is initiated by starvation. The complete sequence of one such developmentally regulated KRP (designated K7) was determined and found to be a novel member of the kinesin superfamily. The motor domain of K7 is most similar to that of conventional kinesin, but unlike conventional kinesin, K7 is not predicted to have an extensive ␣-helical coiled-coil domain. The nonmotor domain is unusual and is rich in Asn, Gln, and Thr residues; similar sequences are found in other developmentally regulated genes in Dictyostelium. K7, expressed in Escherichia coli, supports plus end-directed microtubule motility in vitro at a speed of 0.14 m/s, indicating that it is a bona fide motor protein. The K7 motor is found only in developing cells and reaches a peak level of expression between 12 and 16 h after starvation. By immunofluorescence microscopy, K7 localizes to a membranous perinuclear structure. To examine K7 function, we prepared a null cell line but found that these cells show no gross developmental abnormalities. However, when cultivated in the presence of wild-type cells, the K7-null cells are mostly absent from the prestalk zone of the slug. This result suggests that in a population composed largely of wild-type cells, the absence of the K7 motor protein interferes either with the ability of the cells to localize to the prestalk zone or to differentiate into prestalk cells. INTRODUCTIONSince the prototype kinesin was purified from squid axoplasm (Lasek and Brady, 1985;Schroer et al., 1985;Vale et al., 1985), dozens of related proteins (here referred to as kinesins or kinesin-related proteins [KRPs]) have been identified in organisms ranging from fungi to mammals (Moore and Endow, 1996). KRPs share a conserved mechanochemical motor domain responsible for ATP hydrolysis and microtubule binding. Outside of the 350-amino acid-long motor domain, KRPs show great sequence diversity, but regions predicted to mediate homodimerization via formation of coiled-coil structures are often present (Goldstein, 1993). Sequence diversity among KRPs outside of the motor domain is thought to play a key role in their functional diversity by specifying which proteins and cargo associate with the motor (Goldstein, 1993). Kinesins have been shown to drive the movement of vesicles in fast axonal transport (Hall and Hedgecock, 1991;, the movement of vesicles between the Golgi † Corresponding Author. E-mail address: hostos@bioc.rice.edu. and the ER , and the movement of other organelles, including secretory vesicles, lysosomes, mitochondria, and nuclei (

Microtubule-Nucleus Interactions in Dictyostelium discoideum Mediated by Central Motor Kinesins

“…Since Dictyostelium possesses a relatively small number of motor proteins (13 kinesin, 1 dynein, and 13 myosin isoforms [23,24,26]), it combines advantages of terrific cytology with straightforward molecular genetics and thus represents an excellent model to investigate individual and combined motor protein actions. To date, 11 of the 13 kinesin motors have been analyzed functionally (5,17,18,30,42,46,51,60). Only 1 of these 11 motors, Kif3, a member of the kinesin-1 family of organelle transporters, appears to be essential for organism viability (51).…”

A Novel Actin-bundling Kinesin-related Protein from Dictyostelium discoideum

“…In addition, some KRPs were recently identified as motors associating with membranous structures in Dictyostelium. One of them, K7, was shown to be involved in the development of Dictyostelium cells (6). DdUnc104, the closest relative of Unc104/KIF1 KRPs in Dictyostelium, was purified using an organelle transport assay and shown to participate in membrane transport in cells (7).…”

Identification and phylogenetic analysis of Dictyostelium discoideum kinesin proteins

“…1, Table 1). Five of the sequences have already been published [30-33] as well as fragments of Dd Kif3, Dd Kif4, Dd Kif6, and Dd Kif8 [30]. Since genes belonging to the same family should be designated by using the same acronym, we decided to give all the kinesins the acronym Kif followed by a number.…”

“…For example, in Dictyostelium cells, the levels of DPYK1, a SAM-containing protein-tyrosine kinase, increase as the cells initiate their signal-mediated developmental cycle [64]. The SAM domain of Dd Kif6 might therefore be the reason for its observed developmental regulation [30]. …”

“…Already in the early days of kinesin research the first Dictyostelium kinesin was isolated from cell lysates as a 105 kDa protein, based on its nucleotide-dependent binding to microtubules [29]. About ten years later, a PCR-based approach resulted in the full-length sequence of one kinesin (K7) and short fragments of further kinesins (K3, K4, K6, and K8) [30]. K7 was also analysed by immunofluorescence microscopy and shown to localise to a membranous perinuclear structure.…”