Figure 1.
HMT-1 forms oligomeric complexes.
A. SDS/PAGE and Western blot analyses of HMT-1::GFP. Aliquots (30 µg/lane) of total (Total), soluble (Soluble) or microsomal membrane proteins (Membr) isolated from worms expressing either HMT-1::GFP (HMT-1-GFP) or GFP alone (GFP) under the control of the hmt-1 promoter were subjected to SDS-PAGE and immunoblot analysis. B. Membrane proteins isolated from HMT-1::GFP expressing worms were solubilized either with PFO (PFO-solubilization) or with SDS (SDS-solubilization) and separated by FPLC on Superose 6HR column. Fractions (250 µl) were collected, proteins were precipitated with TCA, and HMT-1::GFP was detected by SDS/PAGE and Western analysis. C. The apparent molecular masses of PFO- or SDS- extracted HMT-1::GFP after FPLC analysis were estimated based on the linear regression of the retention time of molecular mass markers.
Figure 2.
A. Schematic representation of the topology of the full-length HMT-1 polypeptide. Based on the predicted topology (TMHMM software, version 2.0), the NH2-terminus is located outside (Lumen), whereas the COOH-terminus is inside (Cytosol). Also shown are the HMT-1 core region (ABC core) consisting of a single transmembrane domain (TMD1) with six transmembrane spans and a single nucleotide binding domain (NBD1). In addition to a core region, HMT-1 possesses the N-terminal extension (NTE), comprised of a membrane spanning domain (TMD0) and a linker domain (L0). B. Full-length HMT-1 was fused at the C-terminus with CubPLV (HMT-Cub) or with NubG at the C- or N-termini (NubG-HMT and HMT-NubG, respectively). The orientation of CubPLV and NubG is based on the predicted topology of HMT-1. C. Protein-protein interactions of HMT-1 as detected by mbSUS. Growth conditions are indicated below each panel; concentrations of yeast cells are indicated on the left. Numbers across the top represent experiments and controls as follows. Interaction tests where HMT-1-CubPLV was used as bait: 2, HMT-Cub + NubG-HMT; 3, HMT-Cub + HMT-NubG; 7, HMT-Cub + KAT1-NubG. Controls for self-activation: 1, HMT-Cub + NubG; 4, Cub + HMT-NubG. Interaction assays using AtKAT1-CubPLV as bait: KAT1-Cub + HMT-NubG (negative control); 6, KAT1-Cub + KAT1-NubG (positive control, [27]); 7, HMT-Cub + KAT1-NubG (negative control). Shown are representative results of at least three biological replicates. (SC = synthetic complete medium; Met = methionine; Ade = adenine; His = histidine). D. HMT-1-Nub confers Cd tolerance. Serial dilutions of yeast expressing pNub or HMT-NubG were as indicated. Concentrations of CdCl2 in µM are indicated below each experiment. Note the striking growth difference at 75 µM.
Figure 3.
NTE is essential, but not sufficient for protein-protein interactions of HMT-1.
A. Full-length HMT-1 was fused at the C-terminus with CubPLV (HMT-Cub) or with NubG (HMT-NubG). HMT-1 lacking NTE was fused at the C terminus with NubG (ΔNTE-NubG). B. SDS-PAGE and immunoblot analysis of microsomal membranes prepared from THY.AP5 cells expressing the NubG-fused full-length HMT-1 (HMT-NubG), NubG-fused HMT-1 lacking NTE (ΔNTE-NubG), NTE-fused to NubG (NTE-NubG), or empty NubG vector (NubG). C. Interactions were manifested by the ability of cells to grow on SC media without adenine and histidine (SC), but not in SC media with methionine (SC + Met). Minimal growth relative to the negative controls (co-expression with vector alone, NubG) was observed when HMT-1-NubG prey construct lacking NTE (ΔNTE-NubG) was co-expressed with the full-length HMT-1-CubPLV bait (HMT-Cub). Diploid cells did not grow when the NTE domain fused to NubG (NTE-NubG) was co-expressed with HMT-1-CubPLV bait (HMT-Cub). Interactions were also visualized by the presence of β-galactosidase activity. Note the presence of β-galactosidase activity in cells co-expressing the full-length HMT-1-NubG and HMT-1-CubPLV, but not the full-length HMT-1-CubPLV with ΔNTE-NubG, or NTE-NubG or NubG only.
Figure 4.
NTE is essential, but not sufficient for the ability of HMT-1 to confer Cd tolerance.
A. mbSUS analysis of ΔHMT-1 self-association. Growth conditions are indicated on the left. Numbers across the top represent experiments and controls as follows: 1 - HMT-CubPLV + NubG; 2 – HMT-CubPLV + HMT-NubG; 3 - HMT-CubPLV + ΔNTE-NubG; 4 - ΔNTE-CubPLV + ΔNTE-NubG. Note the presence of cell growth and β-galactosidase activity in cells co-expressing the full-length HMT-1-NubG with HMT-1-CubPLV, but not ΔNTE-NubG with ΔNTE-CubPLV, or the full-length HMT-1-CubPLV with ΔNTE-NubG, or NubG only B. Dilution series of THY.AP5 cells expressing the proteins indicated at the top of the figure grown at the indicated concentrations of CdCl2. See legend to Figure 3 for the identity of the constructs. Concentrations of CdCl2 in µM are indicated below each experiment. Note the striking growth difference at 75 and 100 µM CdCl2.
Table 1.
List of C. elegans strains.
Table 2.
The primer sequences used to clone the full-length and truncated CeHMT-1.