Preparation of physiologically active inside-out vesicles from plant inner mitochondrial membranes.

For many metabolites, the major barrier between cytosol and mitochondrial matrix is the inner membrane of mitochondria, the site of the respiratory electron transport chain. In consequence, it houses numerous transporters which facilitate the controlled exchange of metabolites, ions, and even proteins between these cellular compartments. While their import into the organelle can be studied with isolated mitochondria or mitoplasts, the analysis of their export from the matrix into the intermembrane space or even the cytosol demands for more sophisticated approaches.

Fluorescent Protein-Based Approaches for Subcellular Protein Localization in Plants.

Fluorescent proteins (FPs) remarkably advanced the study of cellular biology of plants. The most common application is their use as reporter proteins to determine the subcellular localization of a protein of interest (POI) by endogenous expression of a suitable FP-POI fusion construct in plant cells. In this chapter we describe three approaches, namely, particle bombardment, protoplast transformation, and Agrobacterium infiltration, to transiently express such fusion constructs in plant cells of different species.

Dual targeting of TatA points to a chloroplast-like Tat pathway in plant mitochondria.

The biogenesis of membrane-bound electron transport chains requires membrane translocation pathways for folded proteins carrying complex cofactors, like the Rieske Fe/S proteins. Two independent systems were developed during evolution, namely the Twin-arginine translocation (Tat) pathway, which is present in bacteria and chloroplasts, and the Bcs1 pathway found in mitochondria of yeast and mammals. Mitochondria of plants carry a Tat-like pathway which was hypothesized to operate with only two subunits, a TatB-like protein and a TatC homolog (OrfX), but lacking TatA.

WHIRLY2 plays a key role in mitochondria morphology, dynamics, and functionality in .

WHIRLY2 is a single-stranded DNA binding protein associated with mitochondrial nucleoids. In the mutant of , a major proportion of leaf mitochondria has an aberrant structure characterized by disorganized nucleoids, reduced abundance of cristae, and a low matrix density despite the fact that the macroscopic phenotype during vegetative growth is not different from wild type. These features coincide with an impairment of the functionality and dynamics of mitochondria that have been characterized in detail in wild-type and mutant cell cultures.

Dual or Not Dual?-Comparative Analysis of Fluorescence Microscopy-Based Approaches to Study Organelle Targeting Specificity of Nuclear-Encoded Plant Proteins.

Plant cells are unique as they carry two organelles of endosymbiotic origin, namely mitochondria and chloroplasts (plastids) which have specific but partially overlapping functions, e. g., in energy and redox metabolism.

Rather rule than exception? How to evaluate the relevance of dual protein targeting to mitochondria and chloroplasts.

Dual targeting of a nuclearly encoded protein into two different cell organelles is an exceptional event in eukaryotic cells. Yet, the frequency of such dual targeting is remarkably high in case of mitochondria and chloroplasts, the two endosymbiotic organelles of plant cells. In most instances, it is mediated by "ambiguous" transit peptides, which recognize both organelles as the target.