Figure 1.
Schematic showing relative size and domain organization of a sample of UCS proteins.
UCS proteins are a highly conserved family of proteins by virtue of the C-terminal myosin motor-binding UCS domain (green). Higher eukaryotes have a TPR domain (pink) at the N-terminus, whereas fungal homologs (e.g. S. cerevisiae She4p, S. pombe Rng3p, and P. anserine Cro1p) lack this TPR domain. The central domain (yellow) is the most variable domain between family members. The H. sapiens UCS protein included here is the striated muscle isoform.
Figure 2.
Isolating Myo2p in the absence of functional Rng3p.
Full-length over-expressed Myo2p motors were purified from wild-type (rng3+ nmt41prom-myo2) and mutant (rng3-65 nmt41prom-myo2) cells following growth at the permissive (25°C) or restrictive (37°C) temperature. A) Western blot depicting Myo2p levels (upper panel) from whole cell lysates following induction of Myo2p over-expression (upon removal of thiamine, time zero) in rng3-65 cells. Cells were shifted to the restrictive temperature at 16 hours post-induction, before any appreciable Myo2p expression is detected by antibody (upper blot). The lower blot shows loading controls (Hsp90 levels) for each sample. Protein samples were normalized based on total protein concentration of the lysates. The plot shown below tracks relative levels of Myo2p detected versus time (post-induction). Relative density values for the Myo2p bands were corrected using densities from accompanying Hsp90 bands; final values were normalized whereby 1 is equal to the maximum value. B) Western blots to determine the relative levels of Myo2p produced in the different isolations. Blots were employed (instead of Coomassie staining) to clearly distinguish the Myo2p heavy chain bands in these crude samples. Upper blot: α-Myo2p depicting the relative levels of Myo2p 1-step purified from cells shifted to 37°C (or retained at the permissive growth temperature in accompanying controls) at 16 hours post-induction. The levels shown are from samples harvested at 28 hours post-induction. Samples were diluted 100-fold prior to loading on gels. Lower blot: loading control showing a non-specific band detected by α-Hsp90 antibodies in undiluted samples. Myo2p concentrations were equalized based on the relative levels observed in these blots (prior to testing of the different samples in motility assays; Table 1).
Table 1.
Motility rates for Myo2p purified with and without Rng3p function.
Figure 3.
The myo2-E1 mutation targets a highly conserved residue in the myosin motor domain.
A) Amino acid sequence alignment (centered on Gly-345 of Myo2p) of a number of different myosins. The alignment shows that the glycine residue (pink bar) corresponding to the amino acid substitution in myo2-E1 (G345R) is a highly conserved residue found throughout the myosin super-family. Hs: Homo sapiens, Dm: Drosophila melanogaster, Dr: Danio rerio, Ce: Caenorhabditis elegans, Sc: Saccharomyces cerevisiae, Sp: Schizosaccharomyces pombe. Skeletal and smooth refer to muscle myosin-IIs; Unc-54 is a myosin-II from C. elegans. B) Homology model of the wild-type Myo2p motor based on the 1br4 crystal structure of smooth muscle myosin-II [53]. Insets highlight the area around the site of the -E1 mutation (G345R) in wild-type and myo2-E1-based structures. The potential introduction of a steric clash between Arg-345 and a conserved tyrosine (Tyr-297) is shown in the myo2-E1 inset.
Figure 4.
Myo2-E1p motors lack activity and stability.
The function of S1 fragments of wild-type Myo2p and -E1 motors were compared in vitro following their over-expression and isolation from fission yeast. A) Histogram comparing the distribution of motility rates of S1-GFP constructs in actin filament gliding assays. Average values are shown inset (0.43±0.05 µm/s for wild-type; 0.01±0.02 µm/s for -E1). B) The ATPase activity of S1-FLAG constructs was assayed and compared over a range of temperatures in the absence of actin and the presence of high salt (0.5 M KCl) with 10 mM CaCl2. (n = 3). C) S1-FLAG proteins were subjected to limited proteolysis by trypsin and then run on SDS-PAGE gels. The results of three independent proteolysis experiments are shown. Samples were taken at 0, 1, 2, and 5 minutes following the addition of trypsin before proteolysis was stopped by the addition of SDS-PAGE sample buffer. The far left lane in the top gel shows the running position of 75 and 50 KDa molecular weight standards, bands which run at the same position as the undigested S1 heavy chain band and its primary breakdown product respectively. The intensity of the Coomassie-stained undigested S1 (75 KDa band) and its breakdown product (50 KDa band) were quantified by densitometry. Signals were normalized by setting the intensity of the 75 KDa band to 1.0 and the 50 KDa band to 0 at the 0 min time point. The plot shows the average densitometry values for both the 75 KDa and 50 KDa bands from the three experiments.
Figure 5.
Unconventional fission yeast myosins Myo1p and Myo52p do not require Rng3p for function.
Myo1p localization and patch lifetimes (A, B) and Myo52p localization and directed intra-cellular motility (C–E) were assayed. A) Myo1p-GFP patch localization in representative wild-type and rng3-65 cells is shown following a 5 hour period of growth at 37°C to attenuate Rng3p function in the mutant. White squares in the main images indicate patches whose lifetimes are charted by the time-lapse images shown in the montages below (0–18 s). Bars: 1 µm. B) Average Myo1p patch lifetimes from wild-type and rng3-65 cells treated as described in A (n = 20). C) Myo52p-3xGFP localization in representative wild-type and rng3-65 cells is shown following a 5 hour period of growth at 37°C to attenuate Rng3p function in the mutant. Bar: 4 µm. D) Plots showing the average Myo52p-3xGFP particle motility rates in wild-type versus rng3-65 cells following 5 hours of growth at 37°C (wild-type, n = 239; rng3-65, n = 218). E) Frequency of Myo52p-3xGFP particle motility (events/cell/2 min movie) following 5 hours of growth at 37°C (from data shown in D).
Figure 6.
Rng3p is required the function of non-essential myosin-II Myp2p.
A Myp2p-head/Myo2p-tail chimera construct was tested for its ability to rescue the growth of temperature-sensitive rng3-65 mutant cells under restrictive growth conditions (36°C). Plasmid transformants were grown under permissive conditions (25°C) on EMM-Leu− Ura− minimal media plates before re-streaking and subsequent incubation on plates at 36°C. Double drop-out plates were employed to accommodate the differing markers of the pGFP-myp2-head/myo2-tail (LEU2) and pGFP-rng3 (ura4+) plasmids. The vector alone transformant (left, negative control) carried empty LEU2 and ura4+ plasmids; the Rng3p transformant (center, positive control) carried an empty LEU2 vector and pGFP-rng3; and the Myp2p-head/Myo2p-tail transformant (right) carried pGFP-myp2-head/myo2-tail and an empty ura4+ vector.
Figure 7.
Destabilizing Myo1p motors leads to recruitment of Rng3p to patch structures.
The -E1 mutation was introduced into myo1 (G308R) in the genome to generate the myo1-E1 strain. A) Representative DIC images of wild-type, rng3-65, myo1-E1, and rng3-65 myo1-E1 cells following growth on YE5S media at 25°C. The double mutant was viable and exhibited morphology defects that were a combination of those observed in each of the single mutants (i.e. cells were elongated like rng3-65 cells and somewhat swollen like myo1-E1 cells). B) Myo1p (via its light chain Cam2p) and Rng3p localization in wild-type myo1+ (top) and myo1-E1 mutant (bottom) cells grown in YE5S media at 25°C. Left: single plane epi-fluorescence images of Cam2p-mCherry, center: single plane epi-fluorescence images of Rng3p-3xGFP, and right: merged images of Cam2p (red) and Rng3p (green) signals. Bars: 4 µm.
Table 2.
Growth rates for wild-type, rng3-65, myo1-E1, and rng3-65 myo1-E1 cells.