ABSTRACT
Studying transcription machinery assembly in vitro is challenging because of long intrinsically disordered regions present within the multi-modular transcription factors. One example is alcohol dehydrogenase repressor 1 (Adr1p) from fermenting yeast, responsible for the metabolic switch from glucose to ethanol. The role of each individual transcription activation domain (TAD) has been previously studied, but their interplay and their roles in enhancing the stability of the protein is not known. In this work, we designed five unique miniAdr1 constructs containing either TADs I-II-III or TAD I and III, connected by linkers of different sizes and compositions. We demonstrated that miniAdr1-BL, containing only PAR-TAD I+III with a basic linker (BL), binds the cognate DNA sequence, located in the promoter of the ADH2 (alcohol dehydrogenase 2) gene, and is necessary to stabilize the heterologous expression. In fact, we found that the sequence of the linker between TAD I and III affected the solubility of free miniAdr1 proteins, as well as the stability of their complexes with DNA. miniAdr1-BL is the stable unit able to recognize ADH2in vitro, and hence it is a promising tool for future studies on nucleosomal DNA binding and transcription machinery assembly in vitro.
Subject(s)
Alcohol Dehydrogenase/genetics , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Protein Engineering/methods , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Transcription Factors/chemistry , Transcription Factors/metabolism , Alcohol Dehydrogenase/chemistry , Alcohol Dehydrogenase/metabolism , Binding Sites , Cloning, Molecular , DNA, Fungal/metabolism , DNA-Binding Proteins/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Pichia/genetics , Pichia/metabolism , Protein Binding , Protein Domains , Protein Stability , Saccharomyces cerevisiae/genetics , Transcription Factors/genetics , Transcriptional ActivationABSTRACT
Understanding the selectivity of aquaporin (AQP) membrane channels and exploiting their biotechnological potential will require structural and functional studies of wild type and modified proteins; however, expression systems have not previously yielded AQPs in the necessary milligrams quantities. Cell free (CF) systems have emerged in recent years as fast, efficient and versatile technologies for the production of high quality membrane proteins. Here, we establish a convenient method to synthesize large amounts of functional human aquaglyceroporin 3 protein (AQP3), an AQP of physiological relevance conducting glycerol and some small neutral solutes besides water. Milligram amounts of AQP3 were produced as a histidine-tagged protein (hAQP3-6His) in an Escherichia coli extract-based CF system in the presence of the non-ionic detergent Brij-98. The recombinant AQP3 was purified by affinity chromatography, incorporated into liposomes and evaluated functionally by stopped-flow light scattering. Correct protein folding was indicated by the high glycerol and water permeability exhibited by the hAQP3-6His proteoliposomes as compared to empty control liposomes. Functionality of hAQP3-6His was further confirmed by the strong inhibition of the glycerol and water permeability by phloretin and HgCl2, respectively, two blockers of AQP3. Fast and convenient CF production of functional AQP3 may serve as basis for further structural/functional assessment of aquaglyceroporins and help boosting the AQP-based biomimetic technologies.
Subject(s)
Aquaporin 3 , Escherichia coli/chemistry , Glycerol/chemistry , Plant Oils/chemistry , Polyethylene Glycols/chemistry , Aquaporin 3/biosynthesis , Aquaporin 3/chemistry , Aquaporin 3/isolation & purification , Escherichia coli/metabolism , Liposomes/chemistry , Mercuric Chloride/chemistry , Phloretin/chemistry , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/isolation & purificationABSTRACT
OsPIP1;1 is one of the most abundant aquaporins in rice leaves and roots and is highly responsible to environmental stresses. However, its biochemical and physiological functions are still largely unknown. The oocyte assay data showed OsPIP1;1 had lower water channel activity in contrast to OsPIP2;1. EGFP and immunoelectron microscopy studies revealed OsPIP1;1 was predominantly localized in not only plasma membrane but also in some ER-like intracellular compartments in the cells. OsPIP1;1 exhibited low water channel activity in Xenopus oocytes but coexpression of OsPIP2;1 significantly enhanced its water permeability. Stop-flow assay indicated that 10His-OsPIP1;1-reconstituted proteoliposomes had significantly higher water permeability than the control liposomes. Overexpression of OsPIP1;1 greatly altered many physiological features of transgenic plants in a dosage-dependent manner. Moderate expression of OsPIP1;1 increased rice seed yield, salt resistance, root hydraulic conductivity, and seed germination rate. This work suggests OsPIP1;1 functions as an active water channel and plays important physiological roles.
