Lipid Composition of Outer Leaflet of Chloroplast Outer Envelope Determines Topology of OEP7

Abstract

OEP7, a 6.7-kDa outer envelope protein of spinach chloroplasts inserts into the outer envelope of the organelle independent of a classical cleavable targeting signal. The insertion of OEP7 was studied to describe the determinants for association with, integration into, and orientation of the protein in the outer envelope of chloroplasts. The insertion of OEP7 into the membrane is independent of outer membrane channel proteins and can be reconstituted with the use of protein-free liposomes. In situ, the binding of OEP7 to the membrane surface is not driven by electrostatic interaction because reduction of phosphatidylglycerol or phosphatidylinositol did not reduce the association with the liposomes. The positively charged amino acids flanking the transmembrane domain at the C terminus are essential to retain the native N(in)-C(out) orientation during insertion into chloroplasts. OEP7 inserts with reversed orientation into liposomes containing the average lipid composition of the outer envelopes. The native like N(in)-C(out) orientation is achieved by reduction of the phoshpatidylglycerol concentration mimicking the composition of the outer leaflet of the outer envelope of chloroplasts. We conclude that the unique lipid composition of the outer leaflet due to lipid asymmetry of the outer envelope is essential for the correct topology of OEP7.

Chloroplast Omp85 proteins change orientation during evolution

“…1E). This confirmed the established topology of Oep7 with its C terminus being exposed to the cytoplasm (36,37) and proved the validity of this technique for further studies.…”

“…Oep7 has a single helical transmembrane segment (36)(37)(38). When C-terminally fused to the 11 fragment (11 cOEM ), Oep7 was able to recruit 1-10 CYT to the outer envelope membrane, resulting in ringlike fluorescence around the circumference of the chloroplasts (11 cOEM with 1-10 CYT Fig.…”

Arabidopsis TRIGALACTOSYLDIACYLGLYCEROL5 Interacts with TGD1, TGD2, and TGD4 to Facilitate Lipid Transfer from the Endoplasmic Reticulum to Plastids

“…To further explore the association of TGD5 with one of the two chloroplast envelope membranes, we employed in vivo chloroplast import and protease protection assays (Xu et al, 2005). For this purpose, TGD5-GFP; four envelope-targeted control proteins, TGD1-HA, TGD2-HA, HA-TGD4 (Xu et al, 2005;Awai et al, 2006;Wang et al, 2012), and OEP7-GFP (Schleiff et al, 2001); and one stromal protein, ATS1-HA (Xu et al, 2005), were transiently expressed in tobacco (Nicotiana tabacum) leaves via Agrobacterium tumefaciens infection. Intact chloroplasts isolated from leaves expressing the transgenes were then treated with thermolysin, which is unable to penetrate the outer envelope or trypsin, which is able to penetrate the outer but not the inner membrane.…”

The role of lipids in the biogenesis of integral membrane proteins

“…The N-terminal seven transmembrane domains of LacY switch their membrane orientation in the absence of PE (adapted from Dowhan et al 2004) virus large envelope protein (Lambert and Prange 2001). On the other hand, OEP7, an envelope protein of chloroplasts, orients with native topology in liposomes that reflect the in vivo lipid composition, but with an opposite topology in liposomes of nonnative composition (Schleiff et al 2001). Given the fact that proteins can assume alternative topologies in wild-type cells, and that lipid mutants display alternative topologies of some integral membrane proteins, it seems conceivable that the lipid composition of the bilayer or transport of the protein to a different subcellular membrane may serve to modulate protein topology and, thereby, protein function.…”