A role for myosin-I in actin assembly through interactions with Vrp1p, Bee1p, and the Arp2/3 complex

Abstract

Type I myosins are highly conserved actin-based molecular motors that localize to the actin-rich cortex and participate in motility functions such as endocytosis, polarized morphogenesis, and cell migration. The COOH-terminal tail of yeast myosin-I proteins, Myo3p and Myo5p, contains an Src homology domain 3 (SH3) followed by an acidic domain. The myosin-I SH3 domain interacted with both Bee1p and Vrp1p, yeast homologues of human WASP and WIP, adapter proteins that link actin assembly and signaling molecules. The myosin-I acidic domain interacted with Arp2/3 complex subunits, Arc40p and Arc19p, and showed both sequence similarity and genetic redundancy with the COOH-terminal acidic domain of Bee1p (Las17p), which controls Arp2/3-mediated actin nucleation. These findings suggest that myosin-I proteins may participate in a diverse set of motility functions through a role in actin assembly.

Figure 3

Myo3p associates with Bee1p and Vrp1p and participates in actin assembly. (A) Coimmunoprecipitation of Myo3p with Bee1p-HA. Extracts prepared from cells expressing Bee1p-HA and either Myo3p, Myo3p(W1157S), or Myo3pΔAD, were immunoprecipitated with anti-HA. Immunoprecipitated proteins were detected by immunoblot analysis with anti-Myo3p (top) or anti-HA (bottom). (B) The COOH-terminal acidic domain of myosin-I is genetically redundant with the COOH-terminal acidic domain of Bee1p. myo5Δ strains expressing GAL1-MYO3 and carrying either myo3ΔAD, bee1ΔAD, or bee1ΔAD myo3ΔAD, were streaked on galactose medium or glucose medium to assess colony formation. (C) The acidic domain of myosin-I is essential for assembly of filamentous actin structures in cells depleted COOH-terminal acidic domain of Bee1p. bee1ΔAD myo3ΔAD myo5Δ cells expressing GAL1-MYO3 were shifted from galactose to glucose medium and stained with rhodamine-phalloidin at increasing time points to visualize filamentous actin (top). Cells depleted of Myo3p after 28 h of growth in raffinose medium were shifted back into galactose medium, to reinduce GAL1-MYO3, for 2 h (bottom).

Figure 3

Myo3p associates with Bee1p and Vrp1p and participates in actin assembly. (A) Coimmunoprecipitation of Myo3p with Bee1p-HA. Extracts prepared from cells expressing Bee1p-HA and either Myo3p, Myo3p(W1157S), or Myo3pΔAD, were immunoprecipitated with anti-HA. Immunoprecipitated proteins were detected by immunoblot analysis with anti-Myo3p (top) or anti-HA (bottom). (B) The COOH-terminal acidic domain of myosin-I is genetically redundant with the COOH-terminal acidic domain of Bee1p. myo5Δ strains expressing GAL1-MYO3 and carrying either myo3ΔAD, bee1ΔAD, or bee1ΔAD myo3ΔAD, were streaked on galactose medium or glucose medium to assess colony formation. (C) The acidic domain of myosin-I is essential for assembly of filamentous actin structures in cells depleted COOH-terminal acidic domain of Bee1p. bee1ΔAD myo3ΔAD myo5Δ cells expressing GAL1-MYO3 were shifted from galactose to glucose medium and stained with rhodamine-phalloidin at increasing time points to visualize filamentous actin (top). Cells depleted of Myo3p after 28 h of growth in raffinose medium were shifted back into galactose medium, to reinduce GAL1-MYO3, for 2 h (bottom).

Figure 3

Myo3p associates with Bee1p and Vrp1p and participates in actin assembly. (A) Coimmunoprecipitation of Myo3p with Bee1p-HA. Extracts prepared from cells expressing Bee1p-HA and either Myo3p, Myo3p(W1157S), or Myo3pΔAD, were immunoprecipitated with anti-HA. Immunoprecipitated proteins were detected by immunoblot analysis with anti-Myo3p (top) or anti-HA (bottom). (B) The COOH-terminal acidic domain of myosin-I is genetically redundant with the COOH-terminal acidic domain of Bee1p. myo5Δ strains expressing GAL1-MYO3 and carrying either myo3ΔAD, bee1ΔAD, or bee1ΔAD myo3ΔAD, were streaked on galactose medium or glucose medium to assess colony formation. (C) The acidic domain of myosin-I is essential for assembly of filamentous actin structures in cells depleted COOH-terminal acidic domain of Bee1p. bee1ΔAD myo3ΔAD myo5Δ cells expressing GAL1-MYO3 were shifted from galactose to glucose medium and stained with rhodamine-phalloidin at increasing time points to visualize filamentous actin (top). Cells depleted of Myo3p after 28 h of growth in raffinose medium were shifted back into galactose medium, to reinduce GAL1-MYO3, for 2 h (bottom).

Figure 4

Myosin-I SH3 domain binds proline-rich ligands in Bee1p and Vrp1p. (A) Coimmunoprecipitation of Vrp1p-Myc with Bee1p-HA. Extracts prepared from cells expressing Vrp1p-Myc and Bee1p-HA were immunoprecipitated with anti-HA. Immunoprecipitated proteins were detected by immunoblot analysis with anti-Myc (top) or anti-HA (bottom). (B) Coimmunoprecipitation of Myo3p with Vrp1p-HA. Extracts prepared from cells expressing Vrp1p-HA and either Myo3p, Myo3p(SH3-W1157S), or Myo3pΔAD were immunoprecipitated with anti-HA. Immunoprecipitated proteins were detected by immunoblot analysis with anti-Myo3p (top) or anti-HA (bottom). (C) Direct association of myosin-I with Vrp1p and Bee1p. GST, GST-Myo3p-SH3-AD, or GST-Myo3p-SH3(W1157S)-AD bound to glutathione-Sepharose beads was mixed with E. coli extracts containing MBP-Vrp1p (1-200), MBP-Vrp1p (211-437), or MBP-Bee1p (213-222). Bound proteins were detected by immunoblot analysis with antibodies to MBP (top) or GST (bottom).

Figure 4

Myosin-I SH3 domain binds proline-rich ligands in Bee1p and Vrp1p. (A) Coimmunoprecipitation of Vrp1p-Myc with Bee1p-HA. Extracts prepared from cells expressing Vrp1p-Myc and Bee1p-HA were immunoprecipitated with anti-HA. Immunoprecipitated proteins were detected by immunoblot analysis with anti-Myc (top) or anti-HA (bottom). (B) Coimmunoprecipitation of Myo3p with Vrp1p-HA. Extracts prepared from cells expressing Vrp1p-HA and either Myo3p, Myo3p(SH3-W1157S), or Myo3pΔAD were immunoprecipitated with anti-HA. Immunoprecipitated proteins were detected by immunoblot analysis with anti-Myo3p (top) or anti-HA (bottom). (C) Direct association of myosin-I with Vrp1p and Bee1p. GST, GST-Myo3p-SH3-AD, or GST-Myo3p-SH3(W1157S)-AD bound to glutathione-Sepharose beads was mixed with E. coli extracts containing MBP-Vrp1p (1-200), MBP-Vrp1p (211-437), or MBP-Bee1p (213-222). Bound proteins were detected by immunoblot analysis with antibodies to MBP (top) or GST (bottom).

Figure 4

Myosin-I SH3 domain binds proline-rich ligands in Bee1p and Vrp1p. (A) Coimmunoprecipitation of Vrp1p-Myc with Bee1p-HA. Extracts prepared from cells expressing Vrp1p-Myc and Bee1p-HA were immunoprecipitated with anti-HA. Immunoprecipitated proteins were detected by immunoblot analysis with anti-Myc (top) or anti-HA (bottom). (B) Coimmunoprecipitation of Myo3p with Vrp1p-HA. Extracts prepared from cells expressing Vrp1p-HA and either Myo3p, Myo3p(SH3-W1157S), or Myo3pΔAD were immunoprecipitated with anti-HA. Immunoprecipitated proteins were detected by immunoblot analysis with anti-Myo3p (top) or anti-HA (bottom). (C) Direct association of myosin-I with Vrp1p and Bee1p. GST, GST-Myo3p-SH3-AD, or GST-Myo3p-SH3(W1157S)-AD bound to glutathione-Sepharose beads was mixed with E. coli extracts containing MBP-Vrp1p (1-200), MBP-Vrp1p (211-437), or MBP-Bee1p (213-222). Bound proteins were detected by immunoblot analysis with antibodies to MBP (top) or GST (bottom).

Figure 5

Model for the role of yeast myosin-I in Vrp1p-Bee1p-Arp2/3 mediated actin assembly. The Myo3/5p SH3 domain (circle) binds to Vrp1p and Bee1p. Myo3/5p and Bee1p bind and stimulate the nucleating activity of the Arp2/3 complex through their COOH-terminal acidic domains (arrow). The WH2 domains of Bee1p and Vrp1p bind and couple actin monomers to the Arp2/3 complex for efficient actin nucleation. To represent the WH2 domains, actin monomers are shown bound to Bee1p and Vrp1p. The interaction of the Arp2/3 complex with the regulatory acidic domains of Myo3p, Myo5p, and Bee1p is expected to be transient. Activation of the Arp2/3 complex leads to its incorporation into the actin cytoskeleton, either as a cap on the pointed end of an actin filament or attached to the side of a filament, creating cross-links and branches. The head domain of myosin-I (oval) may organize the newly formed filaments, displace filaments to facilitate barbed-ended insertion, or transport the Vrp1p-Bee1p-Arp2/3 actin assembly complex to the barbed end of a growing actin filament.

Figure 2

Interaction of Myo3p with subunits of the Arp2/3 complex. (A) Coimmunoprecipitation of Myo3p with Arc40p and Arc19p. Extracts prepared from cells expressing Arc19p-HA, Arc40p-HA, or no tag control, were immunoprecipitated with anti-HA. Immunoprecipitated proteins were detected by immunoblot analysis with antibodies directed against Myo3p (top) or HA (bottom). (B) Interaction of the Myo3p acidic domain with Arc40p and Arc19p. GST-Myo3p-SH3-AD, GST-Myo3p-SH3, or GST-Myo3p-AD bound to glutathione-Sepharose beads was mixed with yeast extracts prepared from cells expressing HA epitope-tagged Arc19p or Arc40p. Bound proteins were detected by immunoblot analysis with antibodies to HA (top) or GST (bottom). (C) Localization of GFP-tagged Myo3p, Myo3p(SH3-W1157S), and Myo3pΔAD. (D) Coimmunoprecipitation of Myo3p with Arc40p. Extracts prepared from cells expressing Arc40p-HA and Myo3p, Myo3p(W1157S), or Myo3pΔAD were immunoprecipitated with anti-HA. Immunoprecipitated proteins were detected by immunoblot analysis with antibodies directed against Myo3p (top) or HA (bottom).

Figure 2

Interaction of Myo3p with subunits of the Arp2/3 complex. (A) Coimmunoprecipitation of Myo3p with Arc40p and Arc19p. Extracts prepared from cells expressing Arc19p-HA, Arc40p-HA, or no tag control, were immunoprecipitated with anti-HA. Immunoprecipitated proteins were detected by immunoblot analysis with antibodies directed against Myo3p (top) or HA (bottom). (B) Interaction of the Myo3p acidic domain with Arc40p and Arc19p. GST-Myo3p-SH3-AD, GST-Myo3p-SH3, or GST-Myo3p-AD bound to glutathione-Sepharose beads was mixed with yeast extracts prepared from cells expressing HA epitope-tagged Arc19p or Arc40p. Bound proteins were detected by immunoblot analysis with antibodies to HA (top) or GST (bottom). (C) Localization of GFP-tagged Myo3p, Myo3p(SH3-W1157S), and Myo3pΔAD. (D) Coimmunoprecipitation of Myo3p with Arc40p. Extracts prepared from cells expressing Arc40p-HA and Myo3p, Myo3p(W1157S), or Myo3pΔAD were immunoprecipitated with anti-HA. Immunoprecipitated proteins were detected by immunoblot analysis with antibodies directed against Myo3p (top) or HA (bottom).

Figure 2

Interaction of Myo3p with subunits of the Arp2/3 complex. (A) Coimmunoprecipitation of Myo3p with Arc40p and Arc19p. Extracts prepared from cells expressing Arc19p-HA, Arc40p-HA, or no tag control, were immunoprecipitated with anti-HA. Immunoprecipitated proteins were detected by immunoblot analysis with antibodies directed against Myo3p (top) or HA (bottom). (B) Interaction of the Myo3p acidic domain with Arc40p and Arc19p. GST-Myo3p-SH3-AD, GST-Myo3p-SH3, or GST-Myo3p-AD bound to glutathione-Sepharose beads was mixed with yeast extracts prepared from cells expressing HA epitope-tagged Arc19p or Arc40p. Bound proteins were detected by immunoblot analysis with antibodies to HA (top) or GST (bottom). (C) Localization of GFP-tagged Myo3p, Myo3p(SH3-W1157S), and Myo3pΔAD. (D) Coimmunoprecipitation of Myo3p with Arc40p. Extracts prepared from cells expressing Arc40p-HA and Myo3p, Myo3p(W1157S), or Myo3pΔAD were immunoprecipitated with anti-HA. Immunoprecipitated proteins were detected by immunoblot analysis with antibodies directed against Myo3p (top) or HA (bottom).

Figure 2

Interaction of Myo3p with subunits of the Arp2/3 complex. (A) Coimmunoprecipitation of Myo3p with Arc40p and Arc19p. Extracts prepared from cells expressing Arc19p-HA, Arc40p-HA, or no tag control, were immunoprecipitated with anti-HA. Immunoprecipitated proteins were detected by immunoblot analysis with antibodies directed against Myo3p (top) or HA (bottom). (B) Interaction of the Myo3p acidic domain with Arc40p and Arc19p. GST-Myo3p-SH3-AD, GST-Myo3p-SH3, or GST-Myo3p-AD bound to glutathione-Sepharose beads was mixed with yeast extracts prepared from cells expressing HA epitope-tagged Arc19p or Arc40p. Bound proteins were detected by immunoblot analysis with antibodies to HA (top) or GST (bottom). (C) Localization of GFP-tagged Myo3p, Myo3p(SH3-W1157S), and Myo3pΔAD. (D) Coimmunoprecipitation of Myo3p with Arc40p. Extracts prepared from cells expressing Arc40p-HA and Myo3p, Myo3p(W1157S), or Myo3pΔAD were immunoprecipitated with anti-HA. Immunoprecipitated proteins were detected by immunoblot analysis with antibodies directed against Myo3p (top) or HA (bottom).

Figure 1

The COOH-terminal acidic domain of yeast myosin-I shares sequence similarity with the COOH-terminal acidic domains of yeast Bee1p and human WASP. The oval represents the head domain of myosin-I, the circle represents the SH3 domain, and the arrow represents the acidic domain.