Insertion of plastidic β-barrel proteins into the outer envelopes of plastids involves an intermembrane space intermediate formed with Toc75-V/OEP80

The insertion of organellar membrane proteins with the correct topology requires the following: First, the proteins must contain topogenic signals for translocation across and insertion into the membrane. Second, proteinaceous complexes in the cytoplasm, membrane, and lumen of organelles are required to drive this process. Many complexes required for the intracellular distribution of membrane proteins have been described, but the signals and components required for the insertion of plastidic β-barrel-type proteins into the outer membrane are largely unknown. The discovery of common principles is difficult, as only a few plastidic β-barrel proteins exist. Here, we provide evidence that the plastidic outer envelope β-barrel proteins OEP21, OEP24, and OEP37 from pea (Pisum sativum) and Arabidopsis thaliana contain information defining the topology of the protein. The information required for translocation of pea proteins across the outer envelope membrane is present within the six N-terminal β-strands. This process requires the action of TOC (translocon of the outer chloroplast membrane). After translocation into the intermembrane space, β-barrel proteins interact with TOC75-V, as exemplified by OEP37 and P39, and are integrated into the membrane. The membrane insertion of plastidic β-barrel proteins is affected by mutation of the last β-strand, suggesting that this strand contributes to the insertion signal. These findings shed light on the elements and complexes involved in plastidic β-barrel protein import.

Ubiquitin-dependent chloroplast-associated protein degradation in plants

Chloroplasts contain thousands of nucleus-encoded proteins that are imported from the cytosol by translocases in the chloroplast envelope membranes. Proteolytic regulation of the translocases is critically important, but little is known about the underlying mechanisms. We applied forward genetics and proteomics in Arabidopsis to identify factors required for chloroplast outer envelope membrane (OEM) protein degradation. We identified SP2, an Omp85-type β-barrel channel of the OEM, and CDC48, a cytosolic AAA+ (ATPase associated with diverse cellular activities) chaperone. Both proteins acted in the same pathway as the ubiquitin E3 ligase SP1, which regulates OEM translocase components. SP2 and CDC48 cooperated to bring about retrotranslocation of ubiquitinated substrates from the OEM (fulfilling conductance and motor functions, respectively), enabling degradation of the substrates by the 26S proteasome in the cytosol. Such chloroplast-associated protein degradation (CHLORAD) is vital for organellar functions and plant development.

An RK/ST C-Terminal Motif is Required for Targeting of OEP7.2 and a Subset of Other Arabidopsis Tail-Anchored Proteins to the Plastid Outer Envelope Membrane

Tail-anchored (TA) proteins are a unique class of integral membrane proteins that possess a single C-terminal transmembrane domain and target post-translationally to the specific organelles at which they function. While significant advances have been made in recent years in elucidating the mechanisms and molecular targeting signals involved in the proper sorting of TA proteins, particularly to the endoplasmic reticulum and mitochondria, relatively little is known about the targeting of TA proteins to the plastid outer envelope. Here we show that several known or predicted plastid TA outer envelope proteins (OEPs) in Arabidopsis possess a C-terminal RK/ST sequence motif that serves as a conserved element of their plastid targeting signal. Evidence for this conclusion comes primarily from experiments with OEP7.2, which is a member of the Arabidopsis 7 kDa OEP family. We confirmed that OEP7.2 is localized to the plastid outer envelope and possesses a TA topology, and its C-terminal sequence (CTS), which includes the RK/ST motif, is essential for proper targeting to plastids. The CTS of OEP7.2 is functionally interchangeable with the CTSs of other TA OEPs that possess similar RK/ST motifs, but not with those that lack the motif. Further, a bioinformatics search based on a consensus sequence led to the identification of several new OEP TA proteins. Collectively, this study provides new insight into the mechanisms of TA protein sorting in plant cells, defines a new targeting signal element for a subset of TA OEPs and expands the number and repertoire of TA proteins at the plastid outer envelope.

Synthesis and transfer of galactolipids in the chloroplast envelope membranes of Arabidopsis thaliana

Galactolipids [monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG)] are the hallmark lipids of photosynthetic membranes. The galactolipid synthases MGD1 and DGD1 catalyze consecutive galactosyltransfer reactions but localize to the inner and outer chloroplast envelopes, respectively, necessitating intermembrane lipid transfer. Here we show that the N-terminal sequence of DGD1 (NDGD1) is required for galactolipid transfer between the envelopes. Different diglycosyllipid synthases (DGD1, DGD2, and Chloroflexus glucosyltransferase) were introduced into the dgd1-1 mutant of Arabidopsis in fusion with N-terminal extensions (NDGD1 and NDGD2) targeting to the outer envelope. Reconstruction of DGDG synthesis in the outer envelope membrane was observed only with diglycosyllipid synthase fusion proteins carrying NDGD1, indicating that NDGD1 enables galactolipid translocation between envelopes. NDGD1 binds to phosphatidic acid (PA) in membranes and mediates PA-dependent membrane fusion in vitro. These findings provide a mechanism for the sorting and selective channeling of lipid precursors between the galactolipid pools of the two envelope membranes.