The yeast class V myosins, Myo2p and Myo4p, are nonprocessive actin-based motors - PubMed (original) (raw)

The yeast class V myosins, Myo2p and Myo4p, are nonprocessive actin-based motors

S L Reck-Peterson et al. J Cell Biol. 2001.

Erratum in

Abstract

The motor properties of the two yeast class V myosins, Myo2p and Myo4p, were examined using in vitro motility assays. Both myosins are active motors with maximum velocities of 4.5 microm/s for Myo2p and 1.1 microm/s for Myo4p. Myo2p motility is Ca(2+) insensitive. Both myosins have properties of a nonprocessive motor, unlike chick myosin-Va (M5a), which behaves as a processive motor when assayed under identical conditions. Additional support for the idea that Myo2p is a nonprocessive motor comes from actin cosedimentation assays, which show that Myo2p has a low affinity for F-actin in the presence of ATP and Ca(2+), unlike chick brain M5a. These studies suggest that if Myo2p functions in organelle transport, at least five molecules of Myo2p must be present per organelle to promote directed movement.

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Figure 2

Figure 2

Motility of Myo2p, Myo2-66p, and Myo4p. (A) Immunoblot analysis of antibody capture motility chambers containing either anti Myo-2p or nonimmune IgG. Chambers were incubated with S3 supernates from either wild-type (WT) or myo2-66 cells. Only chambers containing anti-Myo2p and wild-type S3 exhibited movement, although the myo2-66p was present in the chamber. No Myo2p was present in the IgG control chamber. (B) Summary of motility results using anti-GFP to adsorb Myo4-GFPp. Immunoadsorbtions were performed from both tagged and untagged strains. Movement was observed only in chambers containing anti-GFP and S3 supernate from Myo4-GFPp–expressing cells.

Figure 1

Figure 1

Actin cosedimentation assays. Myo2p (A), Myo2-66p (B), or chick brain M5a (C) in S3 supernates was incubated with F-actin in the presence or absence of 4.0 mM ATP with or without 50 μM free Ca2+. After centrifugation to pellet, the F-actin, the supernate, and pellet fractions were immunoblotted with antibodies to Myo2p (A and B) or M5a (C). D shows a representative Coomassie blue–stained gel to visualize actin. In contrast to M5a, Myo2p fails to bind to actin in the presence of ATP. The motor mutant Myo2-66p exhibits reduced actin binding activity.

Figure 3

Figure 3

Chick brain M5a is a processive motor when assayed using the antibody capture (from S3) in vitro motility assay. (A) Velocity (μm/s ± SD) as a function of M5a tail antibody concentration. No movement was observed at an antibody concentration of 5 μg/ ml. The solid line is a theoretical curve derived from with the duty ratio, f = 1 (see Materials and Methods). The good fit to this line indicates that a single M5a molecule remains strongly bound to an actin filament for nearly its entire catalytic cycle. (B) Landing rate (n ± standard error, where n is the number of landing events per unit area, per unit time; see Materials and Methods) as a function of antibody concentration. The curve fit, derived from , represents the case where n = 1, i.e., only one motor molecule is required to bind a filament and initiate movement.

Figure 4

Figure 4

Myo2p and Myo4p are nonprocessive class V myosins. (A) Average velocity (μm/s ± SD) is plotted as a function of motor concentration. No movement was seen at a Myo2p concentration of 8.0 molecules/μm2. Myo2p motility data were best fit by the theoretical curve representing a duty cycle, f = 0.2 (solid line), indicating that a Myo2p molecule is only strongly bound to actin for ∼ 20% of its entire catalytic cycle. Also shown are theoretical curves representing duty ratios of 1.0 (similar to chick brain M5a, long dash) and 0.05 (similar to myosin-II, short dash). (B) Landing rate (n ± standard error, where n is the number of landing events per unit area, per unit time) is plotted as a function of motor concentration. The landing rate was best fit with Eq.4 when n = 5 (solid line; see Materials and Methods), indicating that at least five Myo2p molecules are needed to bind and initiate the movement of an actin filament. Also shown are theoretical landing rate fits where n is equal to 1 (a processive motor such as M5a, long dash) or 20 (a highly nonprocessive motor, short dash). (C) Myo4p velocity (μm/s ± SD) as a function of anti-GFP antibody concentration. No movement was observed using an antibody concentration of 5 μg/ml.

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