pAUL: a gateway-based vector system for adaptive expression and flexible tagging of proteins in Arabidopsis.

Determination of protein function requires tools that allow its detection and/or purification. As generation of specific antibodies often is laborious and insufficient, protein tagging using epitopes that are recognized by commercially available antibodies and matrices appears more promising. Also, proper spatial and temporal expression of tagged proteins is required to prevent falsification of results. We developed a new series of binary Gateway cloning vectors named pAUL1-20 for C- and N-terminal in-frame fusion of proteins to four different tags: a single (i) HA epitope and (ii) Strep-tagIII, (iii) both epitopes combined to a double tag, and (iv) a triple tag consisting of the double tag extended by a Protein A tag possessing a 3C protease cleavage site. Expression can be driven by either the 35 S CaMV promoter or, for C-terminal fusions, promoters from genes encoding the chloroplast biogenesis factors HCF107, HCF136, or HCF173. Fusions of the four promoters to the GUS gene showed that endogenous promoter sequences are functional and drive expression more moderately and consistently throughout different transgenic lines when compared to the 35 S CaMV promoter. By testing complementation of mutations affected in chloroplast biogenesis factors HCF107 and HCF208, we found that the effect of different promoters and tags on protein function strongly depends on the protein itself. Single-step and tandem affinity purification of HCF208 via different tags confirmed the integrity of the cloned tags

HCF164 Encodes a Thioredoxin-Like Protein Involved in the Biogenesis of the Cytochrome b(6)f Complex in Arabidopsis

To understand the biogenesis of the plastid cytochrome b(6)f complex and to identify the underlying auxiliary factors, we have characterized the nuclear mutant hcf164 of Arabidopsis and isolated the affected gene. The mutant shows a high chlorophyll fluorescence phenotype and is severely deficient in the accumulation of the cytochrome b(6)f complex subunits. In vivo protein labeling experiments indicated that the mutation acts post-translationally by interfering with the assembly of the complex. Because of its T-DNA tag, the corresponding gene was cloned and its identity confirmed by complementation of homozygous mutant plants. HCF164 encodes a thioredoxin-like protein that possesses disulfide reductase activity. The protein was found in the chloroplast, where it is anchored to the thylakoid membrane at its lumenal side. HCF164 is closely related to the thioredoxin-like protein TxlA of Synechocystis sp PCC6803, most probably reflecting its evolutionary origin. The protein also shows a limited similarity to the eubacterial CcsX and CcmG proteins, which are required for the maturation of periplasmic c-type cytochromes. The putative roles of HCF164 for the assembly of the cytochrome b(6)f complex are discussed

Oligonucleotides used for pAUL vector construction.

<p>Oligonucleotides used for pAUL vector construction.</p

Oligonuleotides used for cloning of target genes.

<p>Oligonuleotides used for cloning of target genes.</p

Schematic illustration of the Gateway compatible pAUL destination vector series, showing expression cassettes.

<p>(A) C-terminal fusion vectors pAUL1-16. Expression is driven by either 2x p<i>35 </i><i>S CaMV</i> or endogenous promoter sequences from <i>A. thaliana</i> (p<i>HCF107</i>, p<i>HCF136</i>, p<i>HCF173</i>): pAUL1-3 and pAUL13 carry <i>p35S CaMV</i>; pAUL4-6 and pAUL14 carry p<i>HCF107</i>; pAUL7-9 and pAUL15 carry p<i>HCF136</i>; pAUL10-12 and pAUL16 carry p<i>HCF173</i>. Protein tags are: 3xHA single tag (pAUL1, 4, 7, 10); Strep-tag<i>III</i> single tag (pAUL13-16); 3xHA/Strep-tag<i>III</i> double tag (pAUL2, 5, 8, 11); and 3xHA/Strep-tag<i>III</i>/ProtA triple tag +3C protease cleavage site (pAUL3, 6, 9, 12). (B) N-terminal fusion vectors pAUL17-20. Vectors carry coding sequences for 3xHA single tag (pAUL17); 3xHA/Strep-tag<i>III</i> double tag (pAUL18); 3xHA/Strep-tag<i>III</i>/ProtA triple tag +3C protease cleavage site (pAUL19); and Strep-tag<i>III</i> single tag (pAUL20).</p

One step and tandem-purification of HCF208.

<p>100 (A) or 200 µg (B, C) chlorophyll aliquots of solubilized membrane proteins were applied for purification. Aliquots of 20 µg chlorophyll from extracts and total amounts of eluates were separated by SDS-PAGE, transferred to a nitrocellulose membrane and immunodecorated with antibodies against the HA tag (Anti-HA-Peroxidase) and ATP-Synthase as a control. (A) One step purification of proteins from wild type and HCF208pAUL1 via the HA epitope and competitive elution. (B) Tandem purification of proteins from wild type and HCF208pAUL2 via Strep-tag<i>III</i> and 3xHA (C) Tandem purification of proteins from wild type and HCF208pAUL3 via ProtA tag +3C protease cleavage and Strep-tag<i>III</i>.</p