Extraction of the P3 pellet with Triton X-100, which solubilizes membranes (i

Extraction of the P3 pellet with Triton X-100, which solubilizes membranes (i.e., SV2; Feany et al., 1992), was performed to determine whether the insoluble fraction of KLPs is associated with membranous vesicles. for dendrite specific motor localization are inadequate. We Taribavirin hydrochloride suggest that a novel kinesin sorting mechanism is used by neurons to localize KIF21B protein to dendrites since its mRNA is restricted to the cell body. (Rowe et al., 1994). The panel consists of 94 F2 progeny from a (C57BL/6J SPRET/Ei) F1 female mated to a SPRET/Ei male and DNA from parental C57BL/6J and for 5 min, 9,000 for 10 min, then centrifuged in a Sorvall 1270 rotor at 100,000 for 1 h. 50 g of total protein from each fraction, as determined using Bio-Rad Dc protein assay kit, was separated on 7.5% polyacrylamide gels and transferred to PVDF membrane (Bio-Rad Laboratories) for Western immunoblotting. P3 pellets were extracted by homogenization with a dounce homogenizer and recentrifuged at 100,000 for 1 h. The pellet was resuspended in the starting volume and equal volumes of the pellet and supernatant were analyzed by Western immunoblotting. Construction of KIF21B Motor Protein A KIF21B motor construct (amino acids 1C750) was generated by PCR with the following primers that contained either a NdeI or XhoI restriction enzyme site (5-CTG GTG CCG GAG CAT ATG GCT GGC CAG GGC, and 3-CGC TTG TAG CTT CTC GAG CTC CCT TTC ATA). The PCR product was cloned into the NdeI and XhoI sites of pET-23b (Novagen Inc.). The construct was introduced into BL21 (DE3) bacteria and cells were grown at 37C until an OD600 1.5 and then induced with 0. 5 mM IPTG overnight at room temperature. Cells were harvested by Taribavirin hydrochloride centrifugation and resuspended in lysis buffer (300 mM NaCl, 50 mM sodium phosphate, 0.5 mM MgCl2, 0.01% NP-40, 10 g/ml soybean trypsin inhibitor, 0.7 l/ml -ME, 1 mM PMSF, 0.1 M ATP, pH 7.4) at 1 g/5 ml. Cells were lysed three times with a French press and then spun for 45 min at 30,000 rpm in Rabbit polyclonal to WAS.The Wiskott-Aldrich syndrome (WAS) is a disorder that results from a monogenic defect that hasbeen mapped to the short arm of the X chromosome. WAS is characterized by thrombocytopenia,eczema, defects in cell-mediated and humoral immunity and a propensity for lymphoproliferativedisease. The gene that is mutated in the syndrome encodes a proline-rich protein of unknownfunction designated WAS protein (WASP). A clue to WASP function came from the observationthat T cells from affected males had an irregular cellular morphology and a disarrayed cytoskeletonsuggesting the involvement of WASP in cytoskeletal organization. Close examination of the WASPsequence revealed a putative Cdc42/Rac interacting domain, homologous with those found inPAK65 and ACK. Subsequent investigation has shown WASP to be a true downstream effector ofCdc42 a 647.5 Sorvall rotor at 4C. KIF21B-HIS protein was isolated by incubating the high speed supernatant with 0.5 ml of Ni-NTAC agarose beads (Qiagen Inc.) for 2 h. The beads were washed three times with lysis buffer Taribavirin hydrochloride supplemented with 25 mM imidazole and 1 M ATP, and protein was eluted with lysis buffer + 200 mM imidazole and 1 M ATP. Protein was concentrated by centrifuging the protein in a Axioplan fluorescence microscope, a cooled CCD, and the MetaMorph software package (SBS backcross panel (see Materials and Methods). The KIF21A gene maps to 39.7 on mouse chromosome 15 (syntenic to human chromosome 8 at 8q24), and KIF21B maps to Taribavirin hydrochloride 64.7 on chromosome 1 (syntenic to human chromosome 2 at 2cen-q21). The unique chromosome locations establish KIF21A and KIF21B as independent genes, but no known mouse mutants or human diseases map close to these chromosomal locations. KIF21A and KIF21B Define a Novel KLP Family that Contains WD-40 Repeats Members of a protein family often share a high degree of amino acid similarity, as well as common protein motifs. A comparison of the core amino acids of the KIF21A and KIF21B motor domains to previously identified KLPs reveals that KIF21A and KIF21B are most similar to each other and a Taribavirin hydrochloride KLP sequence (CET01G1) identified during the genome sequencing project (Fig. ?(Fig.11 B). KIF21A and KIF21B proteins share 61% amino acid sequence identity along their entire length (Fig. ?(Fig.11 A) with the highest identity in the NH2-terminal 25% and COOH-terminal 25% of the proteins. Like true kinesin, KIF21A and KIF21B proteins are comprised of three functional domains: an NH2-terminal head motor domain (1C400), a predicted coiled-coil stalk (data not shown; 400C1,000), and COOH tail (1,000 to end; Fig. ?Fig.22 A). Both proteins have a cluster of negatively charged amino acids of unknown function within their stalk domain and seven consensus WD-40 repeats (van der Voorn and Ploegh, 1992; Neer et al., 1994) in their tails (Fig. ?(Fig.2,2, A and B). WD-40 repeats were first identified in -transducin (Simon et al., 1991), and subsequently have been found in numerous, functionally unrelated proteins and are believed to be involved in proteinCprotein interactions (Fig. ?(Fig.22 B; see reviews van der Voorn and Ploegh, 1992; Neer et al., 1994). Thus, the KIF21 family of KLPs may mediate interactions with their cargoes through these WD-40 domains. Open in a separate window Figure 2 KIF21A and KIF21B protein structures and WD-40 repeat core amino acids. (A) Cartoon depicting sequence motifs and domains in KIF21A and KIF21B. (B) Alignment of WD-40 repeat amino acids of KIF21A and KIF21B. Shaded boxes, WD-40.