Identification of a novel force-generating protein, kinesin, involved in microtubule-based motility

Abstract

Axoplasm from the squid giant axon contains a soluble protein translocator that induces movement of microtubules on glass, latex beads on microtubules, and axoplasmic organelles on microtubules. We now report the partial purification of a protein from squid giant axons and optic lobes that induces these microtubule-based movements and show that there is a homologous protein in bovine brain. The purification of the translocator protein depended primarily on its unusual property of forming a high affinity complex with microtubules in the presence of a nonhydrolyzable ATP analog, adenylyl imidodiphosphate. The protein, once released from microtubules with ATP, migrates on gel filtration columns with an apparent molecular weight of 600 kilodaltons and contains 110-120 and 60-70 kilodalton polypeptides. This protein is distinct in molecular weight and enzymatic behavior from myosin or dynein, which suggests that it belongs to a novel class of force-generating molecules, for which we propose the name kinesin.

Figure 1

Cosedimentation of a Soluble Axoplasmic Translocator with Purified Microtubules in the Presence of AMP-PNP and Its Subsequent Release by ATP Axoplasmic supernatant (S2, lane b) was incubated with purified microtubules (lane a) at 100 μg/ml (with or without 10 mM AMP-PNP) for 10 min at 23°C, and then the microtubules were pelleted at 37,000 × g for 30 min at 4°C. Proteins released by ATP (see details below) from AMP-PNP-treated microtubules (lane d) show a prominent band at 110 kd and also induce microtubule movement (+). Neither this band nor movement (−) is associated with material released by ATP from control (AMP-untreated) microtubules (lane c), or with the microtubule pellets of AMP-PNP-treated (lane f) and untreated samples (lane e) after the ATP release. Conditions for ATP release. After the original centrifugation of microtubules, the supernatant was discarded and microtubules were resuspended in 75 μl MTG buffer with or without 10 mM AMP-PNP and then centrifuged as before. The microtubule pellet was washed with MTG buffer (50 μl), then resuspended in 30 μl MTG plus 5 mM ATP and 0.1 M KCl for 30 min at 23°C to release associated proteins. Microtubules were pelleted as before, the supernatants were collected (lanes c and d), and the microtubules were resuspended in 30 μl of MTG (lanes e and f). The positions of molecular weight standards are noted at left.

Figure 2

Cosedimentation of a Translocator from Squid Optic Lobes with Purified Microtubules in the Presence of AMP-PNP, and Its Subsequent Release by ATP Soluble supernatant from squid optic lobes (S3, lane a) was incubated with 100 μg/ml of purified microtubules (lane b) in the presence or absence of 5 mM AMP-PNP as described in Experimental Procedures. Material released from microtubules by 5 mM ATP and 0.1 M KCl (45 min; 4°C) from AMP-PNP-treated samples (lane f) shows both a prominent band at 110 kd and movement-inducing activity; neither characteristic is associated with the supernatant of the untreated samples (lane e) or the final microtubule pellets resuspended in an equal volume of MTG with 2 mM ATP (lanes c and d) after ATP release. The ATP-released translocator (lane f) was tested for rebinding to microtubules. The sample was diluted 5-fold in MTG (to reduce the concentration of ATP/KCl) and incubated with new microtubules (100 μg/ml) in the presence or absence of 5 mM AMP-PNP (15 min; 23°C). The microtubules were pelleted and washed, and proteins were released with 5 mM ATP and 0.1 M KCl for 45 min at 23°C. Material released from the AMP-PNP-treated sample contained the 110 kd polypeptide (lane h) and also induced movement (+); neither characteristic was prominent in the control (AMP-PNP-untreated) sample (lane g).

Figure 3

ATP- and KCl-Dependent Release of the Translocator from Microtubules S3 supernatant was incubated with microtubules and AMP-PNP as described in Experimental Procedures. Many contaminating proteins (lane a), but not the translocator, were removed by resuspending the MT pellet in 2 ml of MTG buffer containing 10 mM AMP-PNP, centrifuging the microtubules, and removing the AMP-PNP-containing supernatant. The translocator could then be obtained by washing the pellet with MTG, releasing with 1 ml of MTG plus 10 mM ATP/0.1 M KCl (30 min; 23°C), centrifuging the microtubules, and collecting the supernatant that contains movement-inducing activity (lane b). The final microtubule pellet was then resuspended in 1 ml of MTG (lane c). To define further the conditions that release translocator from microtubules, S3 supernatant (8 ml) was incubated with microtubules and AMP-PNP, and the microtubule pellet was washed as described above. Microtubules were then aliquoted into eight 100 μl samples in MTG containing the indicated amounts of ATP and KCl and incubated for 30 min at 23°C (lanes d–k). The microtubules were pelleted, and 50 μl of each supernatant was run per lane. Lanes a–c are from a different experiment than lanes d–k.

Figure 4

Gel Filtration Chromatography of Squid Translocator (A) Microtubule-purified translocator was applied to a Bio-Gel A5m column, and the microtubule-movement-inducing and ATPase activities of the eluant fractions were determined. (B) The polypeptide compositions of fractions 21–45 were analyzed by SDS polyacrylamide gel electrophoresis (60 μl loaded per lane); fraction 30 contains approximately 65 μg/ml of protein. (C) A densitometer scan is shown of a lane from the fraction (30) with the greatest movement-inducing activity from another column. Chromatographic conditions: A 0.75 ml sample of microtubule-purified translocator (which moved microtubules at a 1:20 dilution) was applied to a 1 × 47 cm column equilibrated in 100 mM KCl, 50 mM Tris (pH 7.6), 5 mM MgCI₂, 0.5 mM EDTA, and 1 mM ATP. The column was run at 4°C at 5 cm/hr; 0.75 ml fractions were collected. Excluded (V_o) and included (V_T) volume fractions are noted at top. Microtubule movement was measured by serial dilution as described in Experimental Procedures. The tubulin doublet at 55 kd that streaks across the gel is not typical, but is an artifact of this particular gel.

Figure 5

Hydroxyapatite Chromatography of Squid Translocator Purified by Gel Filtration Samples corresponding to fractions 28–32 from the gel filtration column shown in Figure 4 were pooled and applied to a 1 × 6 cm hydroxyapatite column equilibrated with gel filtration buffer at pH 7.4. Proteins were eluted with a 0–0.3 M phosphate gradient, and 0.5 ml fractions were collected. Microtubule-movement-inducing activity, assayed by serial dilution, correlated with the presence of 110 and 65 kd polypeptides in eluant fractions. Fraction 40 contains approximately 35 μg/ml of protein.

Figure 6

Cosedimentation of a 120 Kilodalton Bovine Brain Polypeptide with Squid Microtubules Depends upon AMP-PNP Bovine brain S3 supernatant was incubated with squid optic lobe microtubules (100 μg/ml) in the absence (lane a) or presence (lane b) of 5 mM AMP-PNP for 15 min at 23°C. Microtubules were pelleted, washed once with PEM-taxol-GTP buffer, resuspended in 1/40 of the original volume with the same buffer containing 5 mM ATP and 0.1 M KCl, and then assayed for movement-inducing activity.

Figure 7

Gel Filtration Chromatography of the Microtubule-Purified Bovine Translocator (A) Microtubule-purified bovine proteins (0.75 ml; purified as described in Experimental Procedures) were run on a Bio-Gel A5m column like that described in Figure 4, yielding the profile of ATPase and movement-inducing activities shown. (B) The polypeptide compositions of fractions 21–37 were analyzed by SDS polyacrylamide gel electrophoresis (40 μl per lane). (C) A densitometer scan of the Coomassie stained gel is shown of the lane corresponding to the fraction with greatest movement-inducing activity (30, which contains approximately 40–50 μg/ml of protein). Void (V_o) and excluded (V_T) volumes are indicated.