ATP-Binding Cassette Transporters: Snap-on Complexes?

ATP-binding cassette (ABC) transporters are one of the largest families of membrane proteins in prokaryotic organisms. Much is now understood about the structure of these transporters and many reviews have been written on that subject. In contrast, less has been written on the assembly of ABC transporter complexes and this will be a major focus of this book chapter. The complexes are formed from two cytoplasmic subunits that are highly conserved (in terms of their primary and three-dimensional structures) across the whole family. These ATP-binding subunits give rise to the name of the family. They must assemble with two transmembrane subunits that will typically form the permease component of the transporter. The transmembrane subunits have been found to be surprisingly diverse in structure when the whole family is examined, with seven distinct folds identified so far. Hence nucleotide-binding subunits appear to have been bolted on to a variety of transmembrane platforms during evolution, leading to a greater variety in function. Furthermore, many importers within the family utilise a further external substrate-binding component to trap scarce substrates and deliver them to the correct permease components. In this chapter, we will discuss whether assembly of the various ABC transporter subunits occurs with high fidelity within the crowded cellular environment and whether promiscuity in assembly of transmembrane and cytoplasmic components can occur. We also discuss the new AlphaFold protein structure prediction tool which predicts a new type of transmembrane domain fold within the ABC transporters that is associated with cation exporters of bacteria and plants.

Inducible expression of human papillomavirus-16 L1 capsomeres in the plastomes of Nicotiana tabacum: Transplastomic plants develop normal flowers and pollen.

Human papillomavirus type-16 (HPV-16) is the major HPV type involved in causing cervical cancer among women. The disease burden is high in developing and underdeveloped countries. Previously, the constitutive expression of HPV-16 L1 protein led to male sterility in transplastomic tobacco plants. Here, the HPV-16 L1 gene was expressed in chloroplasts of Nicotiana tabacum under the control of an ethanol-inducible promoter, trans-activated by nucleus-derived signal peptide. Plants containing nuclear component were transformed with transformation vector pEXP-T7-L1 by biolistic gun. The transformation and homoplasmic status of transformed plants was verified by polymerase chain reaction and Southern blotting, respectively. Protein was induced by spraying 5% ethanol for 7 consecutive days. The correct folding of L1 protein was confirmed by antigen-capture ELISA using a conformation-specific antibody. The L1 protein accumulated up to 3 µg/g of fresh plant material. The L1 protein was further purified using affinity chromatography. All transplastomic plants developed normal flowers and produced viable seeds upon self-pollination. Pollens also showed completely normal structure under light microscope and scanning electron microscopy. These data confirm the use of the inducible expression as plant-safe approach for expressing transgenes in plants, especially those genes that cause detrimental effects on plant growth and morphology.

Chloroplast-based inducible expression of ESAT-6 antigen for development of a plant-based vaccine against tuberculosis.

Mycobacterium tuberculosis causes tuberculosis in humans. The major disease burden of tuberculosis lies in developing countries. Lack of an effective vaccine for adults is one of the major hurdles for controlling this deadly disease. In the present study, 6 kDa early secretory antigenic target (ESAT-6) of M. tuberculosis was inducibly expressed in chloroplasts of Nicotiana tabacum. The expression of ESAT-6 in chloroplasts was controlled by T7 promoter that was activated by nuclear-generated signal peptide. Tobacco plants, containing nuclear component, were transformed via biolistic bombardment with pEXP-T7-ESAT-6 obtained by Gateway® cloning. Transformation and homoplasmic status of transplastomic plants was confirmed by polymerase chain reaction and Southern blotting. Plants were induced for protein expression by spraying with 5% ethanol for 1 day, 3 days, 7 days and 10 days. ESAT-6 protein was detected by immunoblot analysis and maximum protein was obtained for 10 days induced plants that was estimated to accumulate up to 1.2% of total soluble fraction of protein. Transplastomic plants showed completely normal morphology. Transplastomic and untransformed plants became slightly chlorotic upon prolonged exposure to ethanol until 10 days. Taken together, this data could help in the development of an antigen-based subunit vaccine against tuberculosis.