Inner Envelope Membrane Protein Research Articles

Targeting of a polytopic membrane protein to the inner envelope membrane of chloroplasts in vivo involves multiple transmembrane segments.

The inner envelope membrane (IEM) of the chloroplast plays crucial roles in forming an osmotic barrier and controlling metabolite exchange between the organelle and the cytosol. The IEM therefore harbours a number of membrane proteins and requires the import and integration of these nuclear-encoded proteins for its biogenesis. Recent studies have demonstrated that the transmembrane segment of single-spanning IEM proteins plays key roles in determining their IEM localization. However, few studies have focused on the molecular mechanisms by which polytopic membrane proteins are targeted to the IEM. In this study, we investigated the targeting mechanism of polytopic IEM proteins using the protein Cor413im1 as a model substrate. Cor413im1 does not utilize a soluble intermediate for its targeting to the IEM. Furthermore, we show that the putative fifth transmembrane segment of Cor413im1 is necessary for its targeting to the IEM. The C-terminal portion containing this transmembrane segment is also able to deliver Cor413im1 protein to the IEM. However, the fifth transmembrane segment of Cor413im1 itself is insufficient to target a fusion protein to the IEM. These data suggest that the targeting of polytopic membrane proteins to the chloroplast IEM in vivo involves multiple transmembrane segments and that chloroplasts have evolved a unique mechanism for the integration of polytopic proteins to the IEM.

The role of the transmembrane domain in determining the targeting of membrane proteins to either the inner envelope or thylakoid membrane.

Chloroplastic membrane proteins can be targeted to any of three distinct membrane systems, i.e., the outer envelope membrane (OEM), inner envelope membrane (IEM), and thylakoid membrane. This complex structure of chloroplasts adds significantly to the challenge of studying protein targeting to various membrane sub-compartments within a chloroplast. In this investigation, we examined the role played by the transmembrane domain (TMD) in directing membrane proteins to either the IEM or thylakoid membrane. Using the IEM protein, Arc6 (Accumulation and Replication of Chloroplasts 6), we exchanged the stop-transfer TMD of Arc6 with various TMDs derived from different IEM and thylakoid membrane proteins and monitored the subcellular localization of these Arc6-hybrid proteins. We showed that when the Arc6 TMD was replaced with a TMD derived from various thylakoid membrane proteins, these Arc6(thylTMD) hybrid proteins could be directed to the thylakoid membrane rather than to the IEM. Conversely, when the TMD of the thylakoid membrane proteins, STN8 (State Transition protein kinase 8) or Plsp1 (Plastidic type I signal peptidase 1), was replaced with the stop-transfer TMD of Arc6, STN8 and Plsp1 were halted at the IEM. From our investigation, we conclude that the TMD plays a critical role in targeting integral membrane proteins to either the IEM or thylakoid membrane.

Determinants for stop‐transfer and post‐import pathways for protein targeting to the chloroplast inner envelope membrane

Chloroplast biogenesis relies on the proper transport of thousands of nuclear encoded proteins into this organelle. The majority of nucleus‐encoded chloroplast proteins are imported via the TOC‐TIC translocons in the chloroplast envelope. Several of these proteins are further targeted to suborganellar compartments by additional targeting systems downstream of the import apparatus. The thylakoid targeting systems were presumably conserved from the original endosymbiont. In contrast, our knowledge of the mechanisms of protein targeting and integration at the inner envelope membrane (IM) is very limited. Previous reports have shown that IM proteins are transferred to the inner membrane via a stop‐transfer mechanism. Recent studies have demonstrated that one component of the Tic machinery is imported into the stroma and re‐inserted into the IM in a post‐import mechanism. This led us to investigate these pathways in more detail. Our analyses confirm the existence of both stop‐transfer and post‐import mechanisms of protein targeting to the IM. Furthermore, our results point to the conclusion that the transmembrane helices are not interchangeable and not sufficient for the choice of the insertion pathway. Further deletion and substitution analyses are being performed to better understand the features that determine protein insertion and topology into the chloroplast IM. Supported by NIH, CAPES/Fulbright.

Arabidopsis Tic40 Expression in Tobacco Chloroplasts Results in Massive Proliferation of the Inner Envelope Membrane and Upregulation of Associated Proteins

The chloroplast inner envelope membrane (IM) plays essential roles in lipid synthesis, metabolite transport, and cellular signaling in plants. We have targeted a model nucleus-encoded IM protein from Arabidopsis thaliana, pre-Tic40-His, by relocating its expression from the nucleus to the chloroplast genome. Pre-Tic40-His was properly targeted, processed, and inserted. It attained correct topology and was folded and assembled into a TIC complex, where it accounted for up to 15% of the total chloroplast protein. These results confirm the existence of a novel pathway for protein targeting to the IM. Tic40-His overexpression resulted in a massive proliferation of the IM (up to 19 layers in electron micrographs) without significant effects on plant growth or reproduction. Consistent with IM proliferation, the expression levels of other endogenous IM proteins (IEP37, PPT, Tic110) were significantly (10-fold) upregulated but those of outer envelope membrane (Toc159), stromal (hsp93, cpn60), or thylakoid (LHCP, OE23) proteins were not increased, suggesting retrograde signal transduction between chloroplast and nuclear genomes to increase lipid and protein components for accommodation of increased accumulation of Tic40. This study opens the door for understanding the regulation of membrane biogenesis within the organelle and the utilization of transgenic chloroplasts as bioreactors for hyperaccumulation of membrane proteins for biotechnological applications.

In vivo quantitative relationship between plastid division proteins FtsZ1 and FtsZ2 and identification of ARC6 and ARC3 in a native FtsZ complex.

FtsZ1 and FtsZ2 are phylogenetically distinct homologues of the tubulin-like bacterial cell division protein FtsZ that play major roles in the initiation and progression of plastid division in plant cells. Both proteins are components of a mid-plastid ring, the Z-ring, which functions as a contractile ring on the stromal surface of the chloroplast IEM (inner envelope membrane). FtsZ1 and FtsZ2 have been shown to interact, but their in vivo biochemical properties are largely unknown. To gain insight into the in vivo biochemical relationship between FtsZ1 and FtsZ2, in the present study we investigated their molecular levels in wild-type Arabidopsis thaliana plants and endogenous interactions in Arabidopsis and pea. Quantitative immunoblotting and morphometric analysis showed that the average total FtsZ concentration in chloroplasts of 3-week-old Arabidopsis plants is comparable with that in Escherichia coli. FtsZ levels declined as plants matured, but the molar ratio between FtsZ1 and FtsZ2 remained constant at approx. 1:2, suggesting that this stoichiometry is regulated and functionally important. Density-gradient centrifugation, native gel electrophoresis, gel filtration and co-immunoprecipitation experiments showed that a portion of the FtsZ1 and FtsZ2 in Arabidopsis and pea chloroplasts is stably associated in a complex of approximately 200-245 kDa. This complex also contains the FtsZ2-interacting protein ARC6 (accumulation and replicatioin of chloroplasts 6), an IEM protein, and analysis of density-gradient fractions suggests the presence of the FtsZ1-interacting protein ARC3. Based on the mid-plastid localization of ARC6 and ARC3 and their postulated roles in promoting and inhibiting chloroplast FtsZ polymer formation respectively, we hypothesize that the FtsZ1-FtsZ2-ARC3-ARC6 complex represents an unpolymerized IEM-associated pool of FtsZ that contributes to the dynamic regulation of Z-ring assembly and remodelling at the plastid division site in vivo.

Reconstitution of protein targeting to the inner envelope membrane of chloroplasts.

The chloroplast envelope plays critical roles in the synthesis and regulated transport of key metabolites, including intermediates in photosynthesis and lipid metabolism. Despite this importance, the biogenesis of the envelope membranes has not been investigated in detail. To identify the determinants of protein targeting to the inner envelope membrane (IM), we investigated the targeting of the nucleus-encoded integral IM protein, atTic40. We found that pre-atTic40 is imported into chloroplasts and processed to an intermediate size (int-atTic40) before insertion into the IM. Int-atTic40 is soluble and inserts into the IM from the internal stromal compartment. We also show that atTic40 and a second IM protein, atTic110, can target and insert into isolated IM vesicles in vitro. Collectively, our experiments are consistent with a “postimport” mechanism in which the IM proteins are first imported from the cytoplasm and subsequently inserted into the IM from the stroma.