Identification of proteins involved in heme transport from plastids into the cytoplasm.

Identification of proteins involved in heme transport from plastids into the cytoplasm.
Heme is an essential cofactor for many metabolic functions, such as redox reactions, gas binding and signal transduction. After synthesis in the plastids within the tetrapyrrole biosynthetic pathway, heme must be transferred through the two plastidal envelope membranes into the cytoplasm to be distributed to the various subcellular compartments. We have recently used a previously described genetically encoded heme sensor1 (HS1) to measure for the first time the relative amounts of free heme in different subcellular compartments of transiently or stably transformed plants. This subcellularly acting sensor is one of the important prerequisites for the intended detection of transport proteins required for plastidic heme export. So far, no heme transporter has been published in plants. In yeasts or mammals, members of the ABC transporter family have already been described as heme transporters. It cannot be excluded that plant heme transport is enabled by a multifactorial transporter, so that the detection of functional heme transport in planta and in vitro remains a challenge until all subunits are identified.
The overall aim of the grant application is to elucidate plastidic heme export and to identify the transport proteins involved. To this end, scientific work is proposed with two objectives. 1. the two most promising candidates selected from the group of ABC transporters will be characterized by a detailed molecular analysis (including the heme content in subcellular compartments) of the transgenic lines with inactivated gene for one of the subunits each. Deregulated protein levels in the whole proteome and interacting proteins involved in heme transport will also be determined. In addition, the subcellularly expressed HS1 in the transgenic lines will be used to detect the subcellular heme pool. 2. in continuation of the suppressor screen of a ferrochelatase1 (FC1) mutant with complementary synthesis of the FC2 isoform, which shows light-sensitive, inhibited root growth due to insufficient heme supply to all cellular compartments outside the plastids, one to two promising mutants will be analyzed biochemically/molecularly after genome sequencing of the isolated suppressor mutants. It is expected that the work program will contribute to the identification of proteins responsible for heme transport into the cytoplasm and heme metabolism in the extraplastidal cell compartments.