Tracking subplastidic localization of carotenoid metabolic enzymes with proteomics

Carotenoids represent a set of pigmented lipids with notable significance to photosynthetic capacity and human health. Their importance has resulted in broad interest in employing metabolic engineering of carotenoid metabolism for enhanced nutritional value and stress resilience of crops. While the enzymatic steps of carotenoid biosynthesis are well defined, the regulation of the reactions for optimized pathway flux remains largely unclear. Attempts at metabolic engineering of carotenoid metabolism, that often result in unexpected metabolic outcomes and difficulty in achieving desired carotenoid levels, highlight the need for a better grasp on the control of carotenoid metabolism to realize rational and predictable engineering. The spatial organization of carotenoid metabolism within the plastid is central to this understanding, however, the localization of enzymes and the nature of their protein-protein interactions remains largely unclear. Concerted effort at investigating the dynamic localizations of carotenoid metabolic enzymes will be crucial in unveiling the regulation of carotenoid metabolism for efficient metabolic engineering. In this chapter, an accessible methodology for the study of protein localization across chloroplast subcompartments is presented. Two alternative approaches for protein analysis, mass spectrometry-based proteomics and immunoblotting, offering parallel and complementary methods are outlined. Furthermore, alternative methods for separation of proteins by denatured or native gel electrophoresis are also presented, allowing additional investigation of protein oligomerization of enzymes.