Novel Substrates of Lyp1 in Saccharomyces cerevisiae: Insights into Alanine and Serine Transport

Growth rate calculations:

The growth of S. cerevisiae Δ10AA pADHXC3GH-LYP1  and S. cerevisiae Δ10AA pADHXC3GH on different amino acids provided as sole nitrogen sources was measured in microplates. The wells in the microplate were filled with culture cells to a final OD600 of 0.04 and YB media supplemented with the amino acid of interest to the desired final concentration (2 mM for studying growth on all amino acids; up to 80 mM for studying growth on increasing concentrations of Ala, Ser, Phe, Val). The prepared microplates included three biological replicates of the strain with the plasmid containing the LYP1 gene and one biological replicate of the strain with the empty vector. For statistical analysis, an additional technical replicate of the strain expressing Lyp1 (including 3 biological replicates) as well as 5 of the vector control were retrieved from (DOI 10.5281/zenodo.8426062) and added to the analysis, resulting in a final of 6 replicates for each sample. The data sets (csv files) are the raw optical density readings from the growth assays (txt files), along with the plate layout metadata (csv files). The growth rates were derived based on the Baranyi growth model, using the growthrates package in R. The null hypothesis that the mean growth rate of S. cerevisiae Δ10AA pADHXC3GH-LYP1 is not different from that of the control S. cerevisiae Δ10AA pADHXC3GH in each of the studied amino acids was tested by using the Student's t-test (p<0.05) (csv file). For describing the dependence of the growth rate on the substrate concentration in the cases of growth on Ala, Ser, Phe and Val, the Monod model was used and the fitting of the respective curves was described in GraphPad Prism (10.4.0) (txt file). 

Plasmid sequences (Genbank files):

pADHXC3GH-LYP1, pADHXC3GH

In vivo transport assays of radiolabeled amino acids:

The regression fitting for the Michaelis-Menten, Lineweaver-Burk and Dixon plots, as well as the calculation of kinetic parameters was performed in GraphPad Prism (10.4.0) (txt files). The null hypothesis that the mean uptake rate of S. cerevisiae Δ10AA pADHXC3GH-LYP1 is not different from the fixed value of the control S. cerevisiae Δ10AA pADHXC3GH in each of the studied amino acids was tested by using the one-sample t-test (p<0.05) in GraphPad Prism (10.4.0) (txt file).

Docking:

For docking Lys, Ala and Ser on the AlphaFold model of truncated Lyp1 (AF-P32487), the AutoDock Vina tool of Chimera (1.17.1) was used. The binding site was defined after structural alignment with AdiC (PDB: 3OB6) and was based on the position of interacting residues of AdiC with its substrate Arg. The docking grid was set to 14x15x17 Å, with the ligand center of mass representing the grid center. The docking application was performed using a globally searching exhaustiveness of 8 and the best 10 derived confirmations were derived (pdbqt and txt files). The hydrogen bonding interaction between the Lyp1 and each docked substrate were obtained in ChimeraX (1.8), with distance tolerance of 0.4 Å and angle tolerance of 20 degrees (txt files).