Plant Plasma Membrane P4 ATPase Figure 4. Expression of ALA1 in yeast requires optimization of the cDNA sequence. (A) Total membrane protein preparations (30 mg) were run on SDS-PAGE gels and subjected to immunodetection with antibodies raised against the engineered epitope tags. Only ALIS proteins and yeALA1 can be detected with the anti-RGSH antibody. (B) ALA1 cDNA was modified to eliminate a putative transcription termination site and five rare codons codifying for Arg (yeALA1) and expressed alone or together with an ALIS in a yeast Ddrs2Ddnf1Ddnf2 deletion mutant. ALA1 was tagged at the N-terminal end with an RGSH10-tag while ALIS proteins bear a RGSH6-tag at the same end. ALA1 and ALA1noTT (ALA1 cDNA codified to eliminate a putative transcription termination site) were also expressed under the same conditions. Total membrane protein preparations (30 mg) were run on SDS-PAGE gels and subjected to immunodetection with antibodies raised against the engineered epitope tags. Bands corresponding to ALIS proteins are immunodecorated with an anti-RGSH antibody, while an anti-HA antibody cannot recognize any band corresponding to HA-tagged ALA1. doi:10.1371/journal.pone.0033042.g004 unknown factor required for protein activity, e.g. a regulatory lipid, an activating kinase and/or other interacting proteins. In conclusion, we have shown that ALA1 localizes to the plasma membrane in plant cells and have a strict requirement for a b-subunit to exit the ER and reach its final location. Materials and Methods In silico Analysis of DNA Sequences Primers were designed on the basis of information contained in the P-type ATPase database (http://traplabs.dk/patbase). The genomic DNA sequence for the ALA1 gene and its adjacent 59UTRs was extracted from The Arabidopsis Information Resource webpage (www.arabidopsis.org). Transcription termina- tion signal predictions were performed online at http://harlequin. jax.org/drupal/?q = yeastRNApred [34]. A graphical codon usage analyzer (http://gcua.schoedl.de/) was used to identify rare codons that might result in low levels of expression of ALA1 in S. cerevisiae. Default parameters were selected in both cases. Two different ALA1 modified versions were designed based in these analyses, ALA1noTT (no transcription termination) and yeALA1 (yeast-enhanced). For ALA1noTT, a predicted transcription termination signal sequence around 2800 bp is removed, while yeALA1 lacks this signal and contains five silent mutations that modify the codon usage for arginine from the plant CGG to the yeast-favoured AGA and AGG. Figure S2 shows a comparison of ALA1 to the mutated versions of the gene. DNA Cloning All Polymerase Chain Reactions (PCRs) carried out in this study were performed using Phusion High-Fidelity DNA polymerase (Finnzymes) under the standard conditions recommended by the manufacturer. In the case of genomic DNA amplification, a 2% final concentration of dimethyl sulfoxide (DMSO) was added to the reaction mixture. A full list of the primers used and their characteristics can be found in Table S1. Table S2 comprises a list of the plasmids obtained by cloning PCR products into commercially available blunt-ended vectors, including a summary PLoS ONE | www.plosone.org 6 April 2012 | Volume 7 | Issue 4 | e33042