Interaction of the endocytic scaffold protein Pan1 with the type I myosins contributes to the late stages of endocytosis

Abstract

The yeast endocytic scaffold Pan1 contains an uncharacterized proline-rich domain (PRD) at its carboxy (C)-terminus. We report that the pan1-20 temperature-sensitive allele has a disrupted PRD due to a frame-shift mutation in the open reading frame of the domain. To reveal redundantly masked functions of the PRD, synthetic genetic array screens with a pan1DeltaPRD strain found genetic interactions with alleles of ACT1, LAS17 and a deletion of SLA1. Through a yeast two-hybrid screen, the Src homology 3 domains of the type I myosins, Myo3 and Myo5, were identified as binding partners for the C-terminus of Pan1. In vitro and in vivo assays validated this interaction. The relative timing of recruitment of Pan1-green fluorescent protein (GFP) and Myo3/5-red fluorescent protein (RFP) at nascent endocytic sites was revealed by two-color real-time fluorescence microscopy; the type I myosins join Pan1 at cortical patches at a late stage of internalization, preceding the inward movement of Pan1 and its disassembly. In cells lacking the Pan1 PRD, we observed an increased lifetime of Myo5-GFP at the cortex. Finally, Pan1 PRD enhanced the actin polymerization activity of Myo5-Vrp1 complexes in vitro. We propose that Pan1 and the type I myosins interactions promote an actin activity important at a late stage in endocytic internalization.

Figure 1.

Pan1 and its C-terminal PRD. (A) Schematic representation of the Pan1 protein illustrating its major protein interaction domains. Under each region is a list of identified protein interactions. The black circles represent Prk1 phosphorylation sites. The C-terminal fragment used in the two-hybrid screen (aa1232–1480) is indicated by the black line. (B) Protein sequence alignment of the PRD of Pan1 and mutant pan1-20. The sequence starts at amino acid 1310 and ends at 1480 for the wild-type and 1488 for the mutant protein. The black line highlights the type I myosin SH3 domain ligand consensus sequence, PXXXPPXXP.

Figure 2.

pan1-20 cells have endocytic defects at permissive temperature. (A) Analysis of Pan1 protein in wild-type and pan1-20 cells. Trichloroacetic acid (TCA) extracts of cells, at 26°C or grown at 26°C and shifted to 37°C for 3 h, were prepared and analyzed by immunoblot analysis with anti-Pan1 antibodies. Each band was quantified and the value relative to wild type grown at 26°C (100%) is reported below each lane. (B) Ste6-GFP localization in mid-log phase wild-type and pan1-20 cells. Bar, 5 μm. (C) Conventional electron microscopy of wild-type (WT) and pan1-20 cells. Arrows indicate invaginations, putative sites of vesicle scission. n, nucleus; v, vacuole; m, mitochondria. Bar, 0.5 μm.

Figure 3.

Synthetic genetic interactions of pan1ΔPRD. (A) The pan1ΔPRD strain was confirmed by immunoblot analysis. TCA lysates from a wild-type and the pan1ΔPRD cells were prepared, and an immunoblot analysis was performed with anti-Pan1 antibodies. (B) Synthetic sick interactions with las17-14 and act1-121. Tetrads resulting from crosses with pan1ΔPRD. (C) Synthetic lethal interaction with sla1Δ. The circles highlight the double mutants. P, parental ditype; NPD, nonparental ditype; T, tetratype. (D) Ste3 stabilization/endocytosis was determined by treating each strain with 5 μg/ml cycloheximide, and removing aliquots at each time. Lysates were probed with rabbit anti-Ste3 antibodies and quantified by chemiluminescent detection.

Figure 4.

The type I myosins Myo3 and Myo3 interact with the PRD of Pan1. (A) Schematic representation of the general domain structure of the type I myosins: with the N-terminal myosin motor domain, three IQ repeats where myosin light chains bind, tail homology (TH) domain 1, TH2, an SH3 domain, and a C-terminal acidic (A) domain. The sequences identified in the yeast two-hybrid clones are represented as bars underneath. (B) His₆ pull-downs. Nickel beads loaded with His₆ or His₆-Pan1 PRD were incubated with lysates of GST, GST-Myo3 SH3, or GST-Myo5 SH3. Bound proteins were detected by immunoblot analysis with antibodies to GST. (C) Reciprocal GST pull-downs. Glutathione beads loaded with GST, GST-Myo3 SH3, or GST-Myo5 SH3 were incubated with lysates of His₆, His₆-Pan1 PRD, or His₆-Pan1 PRDΔ12. Bound proteins were detected by immunoblot analysis with antibodies to His₆. (D) Coimmunoprecipitation of Myo3 and Myo5. Extracts prepared from cells expressing Myo3-myc (left) or Myo5-myc (right) were immunoprecipitated with anti-Pan1. Immunoprecipitated proteins were detected by immunoblot analysis with anti-Myc.

Figure 5.

Spatial and temporal colocalization of Pan1 and Myo5. (A) Single frames from the GFP and RFP channels of a movie and a merged image show the colocalization of Pan1-GFP and Myo5-RFP. Time to acquire one pair was 1.5 s. Bar, 5 μm. (B) Kymograph representation of Pan1-GFP (top) and Myo5-RFP (middle) in a single patch over time. The curvature of Pan1p indicates the patch is moving off the cortex, toward the interior of the cell. (C) Time series showing composition of a single patch over 45 s. The time between each frame is 1.5 s. Top and middle, two separate channels, GFP and RFP respectively; bottom, merged image. (D) Kymographs of Myo5-GFP in wild-type, pan1Δprd, act1-121, pan1Δprd act1-121, las17-14, and pan1Δprd las17-14 cells. Images were collected every second for 120 s. (E) A bar graph indicating the average Myo5-GFP lifetimes from the cells shown in D; n = 20 patches for wild-type and pan1Δprd, n = 10 patches for all others. Asterisk (*) indicates p < 0.0001 in paired Student's t tests for the two strains.

Figure 6.

Pan1 PRD enhances Arp2/3 complex activation by the Myo5p–Vrp1p complex. Representative actin nucleation reactions. Rabbit skeletal muscle actin (2 μM; 5% pyrene labeled) was polymerized with 10 nM Arp2/3 complex, 15 nM Myo5p, 50 nM Vrp1, and 75 nM or 100 nM GST or GST-Pan1 PRD (PRD), when indicated.