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Chloroplast Protein Targeting

A significant unsolved problem in cell biology is understanding how cytoplasmically synthesized proteins are targeted to various compartments within eukaryotic cells.  In plants, chloroplasts offer an excellent system for studying this problem.  Most chloroplastic proteins are encoded in the nucleus, synthesized in the cytoplasm as higher molecular weight precursors, and imported posttranslationally across the two envelope membranes.  The long-term goal of our research program is to understand the molecular details of protein import into chloroplasts and to determine the targeting determinants that direct proteins to various subcellular compartments within the chloroplasts.

Chloroplasts have six distinct sub-compartments to which a protein may be ultimately targeted.  Of these six sub-compartments, chloroplasts contain three different membranes, the outer envelope (OEM), the inner envelope (IEM) and the thylakoid membrane.  Understanding the mechanisms of protein targeting to these various membrane compartments provides a challenging area of research for cell biologists to investigate. The process by which proteins are targeted to the chloroplastic IEM is not well understood.  In general, chloroplastic membrane proteins destined for the IEM are thought to utilize the general import pathway (see Fig. 1), but are then diverted to the IEM via a poorly understood process.  Two pathways have been proposed for the targeting of proteins to the inner envelope membrane: the stop-transfer and conservative sorting pathways (Fig. 1).  In the stop-transfer pathway, transport across the IEM is halted, leading to insertion into this membrane during the import process (Fig. 1A).  In the conservative sorting pathway, import into the stroma is completed before insertion into the IEM occurs as a discrete second step (Fig. 1B). We are investigating several known inner envelope membrane proteins in order to identify the targeting determinants that direct these proteins to use either the stop-transfer or conservative sorting pathway.  Furthermore, chloroplasts offer an added level of complexity in that they contain an additional membrane system that is not present in other organelle types (i.e., peroxisomes, mitochondria, etc.) called the thylakoids. Understanding how proteins discriminate between being routed to either the inner envelope membrane or the thylakoid membrane will add to our knowledge regarding intracellular trafficking pathways within chloroplasts. Our current research efforts are focused on the three specific objectives outlined below.

Figure 1. The (A) Stop-Transfer Pathway and (B) the Conservative Sorting Pathway, hypothesized, to direct chloroplastic proteins to the inner envelope membrane. GIP, general import pathway; OEM, outer envelope membrane; IMS, intermembrane space; IEP, inner envelope membrane; TP, transit peptide; TMD, transmembrane domain.

1. Identifying Critical Regions within the Ser/Pro-rich Domain that Directs Tic40 to the Chloroplastic Inner Envelope Membrane

Tic40 is part of the chloroplastic protein import apparatus that is anchored in the inner envelope membrane by a single N-terminal transmembrane domain and has a topology in which the bulk of the C-terminal domain is orientated toward the stroma. Tic40 uses a multi-step targeting pathway for its insertion into the inner envelope membrane.  Using an in vitro import assay, we have shown that the sorting of Tic40 uses a bipartite transit peptide. The first portion is cleaved by the stromal processing peptidase generating a soluble Tic40 stromal intermediate,  iTic40, that is further processed by a second unknown peptidase, thereby  generating its mature form (mTic40). Using deletion mutants, we have identified a sequence motif N-terminal of the transmembrane domain which is essential for reinsertion of iTic40 into the inner envelope membrane. We have designated this novel region a serine and proline (Ser/Pro)-rich domain (Fig. 2).  We are  investigating various regions (i.e. poly serine and proline clusters) within the Ser/Pro rich-domain in greater detail in order to determine what specific residues and/or structural features within this domain are critical for redirecting and inserting Tic40 into the inner envelope membrane of chloroplasts.  Information derived from our Tic40 investigation should allow us to identify signature targeting sequences that distinguish whether a protein destined for the inner envelope membrane uses the ‘stop-transfer’ or ‘conservative sorting pathway’.

Figure 2. Analysis of the Ser/Pro-rich Domain. Mutagenesis study of the Ser/Pro-rich domain of Tic40. Serine to alanine substitutions made in both the Ser/Pro-rich domain and the TMD of Tic40 are highlighted in red. The conserved proline domain is outlined in blue.

2. Investigating the Multiple Targeting Pathways that Direct Proteins to Various Membrane Systems  within the Chloroplasts

To ensure the efficient and correct targeting of proteins to their final destination, chloroplasts have evolved numerous internal protein routing systems.  We are examining the various sorting mechanisms involved in the targeting of different known proteins to the inner envelope membrane.  Using different model inner envelope membrane proteins (Fig. 3) and standard molecular biology techniques, we are attempting to identify essential targeting determinants that direct these proteins to use either the stop-transfer or conservative sorting pathway (Fig. 1).  Many different biophysical features of an inner envelope membrane protein that could serve as a signature targeting sequence will be considered for this analysis (i.e. size and hydrophobicity of a transmembrane domain; charge distribution; prevalence of prolines and serines near or within a TMD, etc).  Finally, chloroplasts offer an added challenge since they contain an additional membrane system that is not present in other organelle types (i.e. peroxisomes, mitochondria etc.) called the thylakoids. Understanding how proteins discriminate between being routed to either the IEM or the thylakoid membrane will add to our knowledge regarding intracellular trafficking pathways within chloroplasts.  Hence, we are also comparing an inner envelope membrane protein (i.e Tic40) with a well characterized thylakoid membrane protein (i.e.PsbX) in order to identify unique targeting motifs that are responsible for allowing these proteins to discriminate between being routed to either the inner envelope membrane or to the thylakoid membrane, respectively.

Figure 3. Model proteins directed to the inner envelope membrane by multiple targeting pathways

3. Identifying and Characterizing Protein Complexes Located at the Chloroplastic Inner Envelope Membrane

The inner envelope membrane contains numerous protein complexes that are involved in a variety of metabolic functions.  However, identification and characterization of stable IEM complexes has proven to be difficult for a variety of reasons.  Recently, we have finished a pea envelope proteomic project (Fig. 4) that utilized a novel pea protein database generated from 454 sequencing of various isolated pea cDNA libraries (established by the Dr. Andreas Weber, Institut für Biochemie der Pflanzen,  Heinrich-Heine-Universität).  This analysis yielded the identification of approximately 250 candidate IEM proteins and likewise confirmed the usefulness of our protein database that was derived from a non-model plant species (i.e., pea plant).  More importantly, we can now exploit the convenience of obtaining large quantities of IEMs from a non-model plant species (i.e., pea), thus allowing us to isolate complexes from pea IEMs, and to determine the complete protein composition of these complexes by applying various proteomic approaches.  Currently, we are pursing several different cell biology and proteomic strategies in order to characterize protein complexes at the IEM.  Elucidating the composition of different protein complexes anchored to the IEM should provide new information as to the function of these complexes within the chloroplasts.

Figure 4. Strategy to isolate and characterize inner envelope membrane protein complexes.

Selected Publications

Joanna Tripp, Kentaro Inoue, Kenneth Keegstra, John E. Froehlich (2007) A novel serine/proline-rich domain in combination with a transmembrane domain is required for the insertion of AtTic40 into the inner envelope membrane of chloroplasts. Plant J 52: 824-838

Andrea Bräutigam, Roshan P. Shrestha, Doug Whitten, Curt G. Wilkerson, Kevin M. Carr, John E. Froehlich, John B. Ohlrogge, Andreas P.M. Weber (2007) Massively-parallel pyrosequencing of cDNAs enables proteomics in non-model species: Comparative analysis of a species specific database generated by pyrosequencing and non-species specific databases for proteome analysis of pea chloroplast envelopes (Submitted, BioTechniques 2007)

John E. Froehlich, Brett Phinney, Curt Wilkerson, W. Keith Ray, Rosemary McAndrew, Douglas Gage, Katherine Osteryoung (2003) Mass spectrometry based proteomic study of Arabidopsis thaliana chloroplastic envelope membranes utilizing alternatives to traditional two-dimensional electrophoresis. J Proteome Research 2: 413-425

Stanislav Vitha, John E Froehlich, Koksharova O,  Harrie van Erp H, Katherine W Osteryoung (2003) Arabidopsis ARC6 is a J-domain plastid division protein whose prokaryotic ancestors are unique to cyanobacteria. Plant Cell 15: 1918-1933

John E. Froehlich,  Aya Itoh and Gregg Howe (2001) Tomato allene oxide synthase and fatty acid hydroperoxide lyase, two cytochrome P450s involved in oxylipin metabolism, are targeted to different membranes of chloroplast envelope. Plant Physiol 125: 306-317

Shin-ya Miyagishimaa, John E. Froehlich, Katherine W. Osteryoung  (2006) The outer envelope protein PDV1, together with its paralogue PDV2, mediates recruitment of the dynamin-related protein ARC5 to the plastid division site in Arabidopsis.  Plant Cell 18: 2517-2530.

Changcheng  Xu, Jilian  Fan, John E. Froehlich , Awai K, Christoph Benning (2005) Mutation of the TGD1 chloroplast envelope protein affects phosphatidate metabolism in Arabidopsis. Plant Cell 17: 3094-3110

Diane Constan, John E Froehlich, Sowkya Rangarajan, Kenneth Keegstra (2004) A Stromal Hsp100 protein is required for normal chloroplast development and function in Arabidopsis. Plant Physiol 136: 3605-3615

Diane Jackson, John E. Froehlich and Kenneth Keegstra (1998) The topology of Tic 110, a component of the chloroplastic protein import apparatus. J Biol Chem 273: 16583-16588

Pat Tranel, John E. Froehlich Arun Goyal, and Kenneth Keegstra (1995) A component of the chloroplastic protein import apparatus is targeted to the outer envelope membrane via a novel pathway EMBO J14: 2436-2446.

John E. Froehlich, Christoph Benning, and Peter Dörmann (2001) The digalactosyldiacylglycerol synthase DGD1 is inserted into the outer Envelope membrane of chloroplasts in a manner independent of the general import pathway and does not depend on MGDG synthase for DGDG biosynthesis. J Biol Chem 276: 31806-31812

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