ALB3 Insertase Mediates Cytochrome b Co-translational Import into the Thylakoid Membrane.

The cytochrome b f complex occupies an electrochemically central position in the electron-transport chain bridging the photosynthetic reaction center of PS I and PS II. In plants, the subunits of these thylakoid membrane protein complexes are both chloroplast and nuclear encoded. How the chloroplast-encoded subunits of multi-spanning cytochrome b are targeted and inserted into the thylakoid membrane is not fully understood. Experimental approaches to evaluate the cytochrome b import mechanism in vivo have been limited to bacterial membranes and were not a part of the chloroplast environment. To evaluate the mechanism governing cytochrome b integration in vivo, we performed a comparative analysis of both native and synthetic cytochrome b insertion into purified thylakoids. Using biophysical and biochemical methods, we show that cytochrome b insertion into the thylakoid membrane is a non-spontaneous co-translational process that involves ALB3 insertase. Furthermore, we provided evidence that CSP41 (chloroplast stem-loop-binding protein of 41 kDa) interacts with RNC-cytochrome b complexes, and may be involved in cytochrome b (petB) transcript stabilization or processing.