Arp10p is a pointed-end-associated component of yeast dynactin

Abstract

In metazoans, dynein-dependent vesicle transport is mediated by dynactin, containing an actin-related protein, Arp1p, together with a cargo-selection complex containing a second actin-related protein, Arp11. Paradoxically, in budding yeast, models of dynactin function imply an interaction with membranes, whereas the lack of microtubule-based vesicle transport implies the absence of a cargo-selection complex. Using both genetic and biochemical approaches, we demonstrate that Arp10p is the functional yeast homologue of Arp11, suggesting the possible existence of a pointed-end complex in yeast. Specifically, Arp10p interacts with Arp1p and other dynactin subunits and is dependent on Arp1p for stability. Conversely, Arp10p stabilizes the dynactin complex by association with the Arp1p filament pointed end. Using a novel hRAS-Arp1p one-hybrid assay, we show that Arp1p associates with the plasma membrane dependent on dynactin subunits, but independent of dynein, and sensitive to cell wall damage. We directly show the association of Arp1p with not only the plasma membrane but also with a less dense membrane fraction. Based on the hRAS-Arp1p assay, loss of Arp10p enhances the apparent association of dynactin with the plasma membrane and suppresses the loss of signaling conferred by cell wall damage.

Figure 1.

Structural and genetic evidence for Arp10p interaction with the Arp1p filament. (A) Arp10p modeled at the pointed end of Arp1p (gray). Arp10p (blue) shows the position of deleted residues (red) or small insertions (yellow). Deletions are at the pointed end, where they would prevent association of further subunits; insertions are near the heterotypic interface where they could exert stabilizing forces. ARP1 alleles used in this study are indicated in green. (B) Genetic interaction of arp10Δ with ARP1. Growth was examined with kip3-15 which renders ARP1 essential, although in this strain background kip3-15 arp1Δ or kip3Δarp1Δ provide for weak growth at 23°C. kip3-15 and kip3Δ are equivalent under the conditions examined (Clark and Rose, 2005). Deletion of ARP10 exacerbates both and Ts^- (arp1-113) and specific pseudo wild-type alleles (arp1-44, arp1-46), but not others (arp1-37). (C) Deletion of ARP10 increases wild-type ARP1 protein threshold for growth. The ARP1 ORF is driven by a doxycycline-repressible promoter in a kip3Δ TET-SSN6 background. Addition of doxycycline (10 μg/ml) decreases Arp1p levels below the threshold for wild-type growth. Removal of Arp10p further enhances this defect. The unrepressed promoter has slightly lower levels of Arp1p, evident as a reduction of growth in the absence of Arp10p. 1:10 serial dilutions are shown. (D) Deletion of ARP10 abates overexpression rescue of specific ARP1 mutants in kip3-15. Overexpression rescues particular Ts^- and lethal ARP1 mutants. Elimination of Arp10p inhibits remediation of specific alleles.

Figure 2.

Two-hybrid interactions define Arp10p association with the dynactin complex. (A) Individual dynactin subunits were fused to the galactose activating domain (GAD) or DNA binding domain (GBD). Interaction was monitored with a HIS3 reporter at 30°C. A 10-fold serial dilution is shown for each strain. The GBD-ARP10-GAD-JNM1 interaction was only apparent when endogenous JNM1 was deleted. (B) Summary of dynactin complex interactions detectable by two-hybrid. Only Arp10p fails to interact with itself, consistent with a capping protein. In one configuration, endogenous Jnm1p interferes with the Arp10p-Jnm1p interaction. AD, GAL activation domain; BD, GAL DNA binding domain. The budding yeast dynactin complex is shown below with the pointed end to the right, adapted from Schafer et al. (1994). (c) Deletion of ARP10 reduces the interaction of Arp1p-Arp1p, Jnm1p-Jnm1p, and Arp1p-Jnm1p. (D) Pseudo wild-type allele arp1-46 reduces the interaction of Arp10p and Jnm1p with Arp1p, without decreasing the interaction of Arp1p-Arp1p. Note that the more distantly positioned arp1-44, which overlaps with arp1-46, does not weaken this interaction. (E) Pseudo wild-type alleles do not generally reduce protein interactions: arp1-112 reduces the Jnm1p-Arp1p interaction, but not the interaction of Arp10p or Arp1p with Arp1p. HIS, HIS3 reporter.

Figure 3.

Physical interactions of Arp10p. (A) Arp10p protein can be coimmunoprecipitated (IP) with dynactin subunits. IP targets were tagged with HA, whereas the associated protein was tagged with MYC epitope. IPs with anti-HA antibody were detected by immunoblotting with anti-MYC antibody. Control IPs are shown on the right, prepared from strains lacking HA-tagged subunits, but containing MYC-tagged subunits. The molecular weight standards correspond to protein migration on the control blot only. (B) Association of Arp1p and Jnm1p is reduced without Arp10p. Jnm1p was coIP with Arp1p in the presence or absence of Arp10p as in A. Duplicates are shown. Arp1p and Jnm1p are stable without Arp10p (inset). (C) Sedimentation of Arp10-MYCp in the presence and absence of Arp1p, Jnm1p, or both proteins. Cytosols from Arp10-MYCp with or without Arp1p or Jnm1p were examined by rate-zonal sedimentation on 5-20% sucrose gradients. Fractions containing Arp10p were determined with an anti-MYC antibody. Fraction 1 is the bottom of the gradient. Equal volumes of each fraction were loaded in each lane. The lower panel represents the distribution of Arp10p as a fraction of the maximal Arp10p signal. (D) Arp10p is specifically unstable without Arp1p. Arp1p is stable in the absence of Arp10p and other dynein/dynactin subunits. Dynactin subunits Jnm1p and Nip100p are stable without Arp1p, but Arp10p is uniquely unstable without Arp1p. (E) An in vivo assay for Arp10p association with Arp1p. The instability of Arp10p without Arp1p provides a readout of Arp10p association. The steady-state level of Arp10p protein is specifically reduced in those ARP1 mutants showing genetic interaction. (F) Arp10p interaction with ARP1 pseudo wild-type alleles on the Arp1p filament model. The unique residue of arp1-46 and the two residues in common with arp1-44 are positioned such that they could be important for Arp10p contact with two different Arp1p subunits. Note that arp1-44, the weaker of the two alleles in many assays for Arp10p interaction, is more distantly positioned from Arp10p. Arp10p is shown in blue; side chains of Arp10p, absent relative to Arp1p, are not shown. Two underlying Arp1p subunits are shown by wireframe.

Figure 4.

Arp10p interaction with ARP1 pseudo wild-type alleles on the barbed end of Arp1p and rescue of ARP1 alleles by overexpression of Arp10p. (A) Two-hybrid results for both ARP10 and ARP1 interaction with ARP1 barbed-end pseudo wild-type alleles. No ARP1 allele reduces Arp10p-Arp1p interaction, consistent with the association of Arp10p at the pointed, rather than barbed, end of Arp1p. Note that some alleles (arp1-86, -99, -176, and -181) do reduce the Arp1-Arp1 homotypic interaction, demonstrating the allele specificity of the assay. (B) A model of the barbed end of an Arp1p pentamer indicating the position of all barbed-end alanine-scanning alleles, all of which are pseudo wild type. The alleles on only one monomer are labeled owing to the symmetry. (C) Rescue of ARP1 alleles by overexpression of Arp10p from a high-copy plasmid (2μ) was tested in 21 ARP1 alleles, ARP1, and arp1Δ at five temperatures. Only alleles demonstrating rescue are shown.

Figure 5.

Arp1p interaction with the membrane is independent of dynein and is negatively affected by Arp10p. (A) An in vivo Ras-based one-hybrid assay reveals an association between Arp1p and the membrane. A cdc25-2::HPH Ts^- allele in the background of all strains shown eliminates the endogenous Ras activity at 35°C and above. Ras activity can be substituted by a plasmid harboring a heterologous activated Ras allele (hRAS^*) that does not require the nucleotide exchange activity of CDC25. Because hRAS^* lacks residues that would normally direct it to the plasma membrane, it cannot restore Ras activity. Addition of either an N-terminal myristoylation signal or restoration of the C-terminal CAAX sequence to hRAS^* redirects hRAS^* to the plasma membrane, overcoming the loss of endogenous ras activity afforded by cdc25-2, and restoring growth. Fusion of hRAS^* to Arp1p is equally capable of restoring activity. This interaction is not dependent on dynein or its receptor Num1p but is dependent on dynactin subunits Jnm1p and Nip100p. The elimination of Arp10p enhances the interaction, especially evident at 37°C. (B) Arp1p floats in buoyant density gradients with membranes. Isolated membranes were layered under a buoyant density gradient and allowed to reach equilibrium. Twelve 1-ml fractions were taken from the top of the gradient (left in figure) and a sample of each was resolved by SDS-PAGE. A portion of Arp1p floats to the same density as the plasma membrane; however, the majority of Arp1p is found associated with lighter membrane fractions. The position of various membranes was determined by immunoblotting for resident polypeptides. PGK is cytosolic, Pma1p is plasma membrane, Dpm1p is endoplasmic reticulum (ER) membrane, Pep12p is endosomal membrane, and Porin is mitochondrial outer membrane. The ER shows as a low-density membrane fraction due to the inclusion of EDTA.

Figure 6.

hRAS-Arp1, but not RAS-CAAX signaling, is modulated by FKS and ARP10. (A) Introduction of fks1Δ fks1-1154 or fks2Δ into the system does not perturb the activity of myristoylated hRAS^* or hRAS^*-CAAX; however, it eliminates the activity of hRAS^*-ARP1. (B) The further elimination of ARP10 (arp10Δ) remediates the perturbation of hRAS^*-ARP1 caused by fks1Δ fks1-1154, fks2Δ, and fks1Δ alone.