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, 25 (4), 693-700

Structural Basis for Myosin V Discrimination Between Distinct Cargoes

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Structural Basis for Myosin V Discrimination Between Distinct Cargoes

Natasha Pashkova et al. EMBO J.

Abstract

Myosin V molecular motors move cargoes on actin filaments. A myosin V may move multiple cargoes to distinct places at different times. The cargoes attach to the globular tail of myosin V via cargo-specific receptors. Here we report the crystal structure at 2.2 A of the myosin V globular tail. The overall tertiary structure has not been previously observed. There are several patches of highly conserved regions distributed on the surface of the tail. These are candidate attachment sites for cargo-specific receptors. Indeed, we identified a region of five conserved surface residues that are solely required for vacuole inheritance. Likewise, we identified a region of five conserved surface residues that are required for secretory vesicle movement, but not vacuole movement. These two regions are at opposite ends of the oblong-shaped cargo-binding domain, and moreover are offset by 180 degrees. The fact that the cargo-binding areas are distant from each other and simultaneously exposed on the surface of the globular tail suggests that major targets for the regulation of cargo attachment are organelle-specific myosin V receptors.

Figures

Figure 1
Figure 1
Structural overview of the Myo2p globular tail. (A) Ribbon representation of the structure in two orientations (rainbow colors from blue, N-terminus, to red, C-terminus). Vacuole-specific mutations D1297G/N, L1301P, N1304S, and N1307D/N are indicated in magenta. The most amino-terminal residue within the solved structure is 1152. (B) Topology diagram. Subdomain I is blue and subdomain II is red. Cyan indicates the region of the C-loop that is part of subdomain I. All structures presented in the figures were drawn using PyMol (DeLano Scientific LLC).
Figure 2
Figure 2
The vacuole-binding region of the globular tail. (A) Helices H4 and H6 contain the vacuole-specific residues D1297, L1301, N1304, N1307, and Q1233 (magenta). (B) Quantitative analysis of vacuole inheritance of the myo2-Q1233R mutant.
Figure 3
Figure 3
Surface representation of the myosin V globular tail, indicating areas of high sequence conservation. (A) Surface area of the Myo2p globular tail conserved region (shown in orange) predicted to bind to secretory vesicles (Schott et al, 1999). This region of Myo2p is conserved with chicken myosin Va. The sequence in this region is not conserved with Myo4p, a second S. cerevisiae myosin V, which specifically transports mRNA and the peripheral endoplasmic reticulum (Estrada et al, 2003). Blue: subdomain I; red: subdomain II; cyan: vacuole-binding region. (B) Highly conserved residues (ConSurf scale of ‘8'or ‘9') of subdomain I are blue and that of subdomain II in red. Vacuole-specific residues D1297, L1301, N1304, N1307, and Q1233, and the secretory vesicle-specific mutation, Y1415, are indicated.
Figure 4
Figure 4
Identification of the secretory vesicle-binding region. (A) Ribbon representation of subdomain II. (B) Viability assay for secretory vesicle-specific mutants. YEPD plates were incubated at 24 or 37°C. Photographs were taken on day 3.
Figure 5
Figure 5
The vacuole- and secretory vesicle-binding sites are structurally separated within the Myo2p globular tail. Surface residues of subdomain I are shown in blue and that of subdomain II in red. Vacuole-binding site is in cyan and secretory vesicle-specific site is in yellow. This present figure and Figure 3 show the same orientation.

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