Toc64, a New Component of the Protein Translocon of Chloroplasts

A subunit of the preprotein translocon of the outer envelope of chloroplasts (Toc complex) of 64 kD is described, Toc64. Toc64 copurifies on sucrose density gradients with the isolated Toc complex. Furthermore, it can be cross-linked in intact chloroplasts to a high molecular weight complex containing both Toc and Tic subunits and a precursor protein. The 0 Å cross-linker CuCl2 yields the reversible formation of disulfide bridge(s) between Toc64 and the established Toc complex subunits in purified outer envelope membranes. Toc64 contains three tetratricopeptide repeat motifs that are exposed at the chloroplast cytosol interface. We propose that Toc64 functions early in preprotein translocation, maybe as a docking protein for cytosolic cofactors of the protein import into chloroplasts.

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Mechanism of protein import across the chloroplast envelope

Biochemical Society Transactions ◽

10.1042/bst0280485 ◽

2000 ◽

Vol 28 (4) ◽

pp. 485-491 ◽

Cited By ~ 9

Author(s):

K. Chen ◽

X. Chen ◽

D. J. Schnell

Keyword(s):

Protein Import ◽

Essential Elements ◽

Chloroplast Envelope ◽

Envelope Membrane ◽

Outer Envelope ◽

Double Membrane ◽

Protein Subunits ◽

Targeting Signals ◽

Outer Envelope Membrane ◽

Envelope Membranes

The development and maintenance of chloroplasts relies on the contribution of protein subunits from both plastid and nuclear genomes. Most chloroplast proteins are encoded by nuclear genes and are post-translationally imported into the organelle across the double membrane of the chloroplast envelope. Protein import into the chloroplast consists of two essential elements: the specific recognition of the targeting signals (transit sequences) of cytoplasmic preproteins by receptors at the outer envelope membrane and the subsequent translocation of preproteins simultaneously across the double membrane of the envelope. These processes are mediated via the co-ordinate action of protein translocon complexes in the outer (Toe apparatus) and inner (Tic apparatus) envelope membranes.

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Differential accumulation of plastid preprotein translocon components during spruce (Picea abies L. Karst.) needle development

Biological Chemistry ◽

10.1515/bc.2005.091 ◽

2005 ◽

Vol 386 (8) ◽

pp. 777-783 ◽

Cited By ~ 6

Author(s):

Hrvoje Fulgosi ◽

Hrvoje Lepeduš ◽

Vera Cesar ◽

Nikola Ljubešić

Keyword(s):

Protein Import ◽

Protein Translocation ◽

Early Stage ◽

Small Subunit ◽

Outer Envelope ◽

Receptor Isoforms ◽

Evolutionarily Conserved ◽

Functional Complex ◽

Plastid Protein ◽

Preprotein Translocation

Abstract We demonstrate that basic components of the plastid protein-import apparatus originally found in pea, Toc34, Toc159, and Tic110, are also conserved in evolutionarily younger gymnosperms. We show that multiple isoforms of the preprotein receptor Toc34 differentially accumulate in various stages of needle development, while the amounts of Toc159 drastically decrease during chloroplast morphogenesis. Spruce Toc34 and Toc159 receptors are able to recognise and interact with the angiosperm precursor of the Rubisco small subunit. Young proplastids found in closed buds contain a highly elevated number of protein translocation complexes equipped with only two types of outer envelope receptors, Toc159 and a 30-kDa Toc34-related protein. Photosystem II (PSII) can already be assembled in a fully functional complex at this very early stage of needle development, suggesting that no additional receptor isoforms are needed for translocation of all necessary PSII components. We conclude that the accumulation of evolutionarily conserved plastid preprotein translocation components is differentially regulated during spruce needle development.

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A new chloroplast protein import intermediate reveals distinct translocation machineries in the two envelope membranes: energetics and mechanistic implications.

The Journal of Cell Biology ◽

10.1083/jcb.132.1.63 ◽

1996 ◽

Vol 132 (1) ◽

pp. 63-75 ◽

Cited By ~ 53

Author(s):

S V Scott ◽

S M Theg

Keyword(s):

Membrane Transport ◽

Protein Import ◽

Chloroplast Envelope ◽

Envelope Membrane ◽

Outer Envelope ◽

Stromal Compartment ◽

Chloroplast Protein Import ◽

Chloroplast Protein ◽

Outer Envelope Membrane ◽

Envelope Membranes

Chloroplast protein import presents a complex membrane traversal problem: precursor proteins must cross two envelope membranes to reach the stromal compartment. This work characterizes a new chloroplast protein import intermediate which has completely traversed the outer envelope membrane but has not yet reached the stroma. The existence of this intermediate demonstrates that distinct protein transport machineries are present in both envelope membranes, and that they are able to operate independently of one another under certain conditions. Energetic characterization of this pathway led to the identification of three independent energy-requiring steps: binding of the precursor to the outer envelope membrane, outer membrane transport, and inner membrane transport. Localization of the sites of energy utilization for each of these steps, as well as their respective nucleotide specificities, suggest that three different ATPases mediate chloroplast envelope transport.

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

The Plant Cell ◽

10.1093/plcell/koab052 ◽

2021 ◽

Author(s):

Lucia E Gross ◽

Anna Klinger ◽

Nicole Spies ◽

Theresa Ernst ◽

Nadine Flinner ◽

...

Keyword(s):

Membrane Proteins ◽

Protein Import ◽

Membrane Insertion ◽

Intermembrane Space ◽

Envelope Membrane ◽

Outer Envelope ◽

Pea Proteins ◽

Outer Envelope Membrane ◽

Correct Topology ◽

Shed Light

Abstract 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.

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Identification of a human PTS1 receptor docking protein directly required for peroxisomal protein import

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.95.14.8087 ◽

1998 ◽

Vol 95 (14) ◽

pp. 8087-8092 ◽

Cited By ~ 117

Author(s):

M. Fransen ◽

S. R. Terlecky ◽

S. Subramani

Keyword(s):

Protein Import ◽

Peroxisomal Protein ◽

Docking Protein

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The essential yeast protein MIM44 (encoded by MPI1) is involved in an early step of preprotein translocation across the mitochondrial inner membrane.

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7364 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7364-7371 ◽

Cited By ~ 76

Author(s):

J Blom ◽

M Kübrich ◽

J Rassow ◽

W Voos ◽

P J Dekker ◽

...

Keyword(s):

Protein Import ◽

Early Stage ◽

Proteolytic Processing ◽

Early Step ◽

Direct Role ◽

Inner Membrane ◽

Mitochondrial Inner Membrane ◽

Preprotein Translocation ◽

In Transit

The essential yeast gene MPI1 encodes a mitochondrial membrane protein that is possibly involved in protein import into the organelle (A. C. Maarse, J. Blom, L. A. Grivell, and M. Meijer, EMBO J. 11:3619-3628, 1992). For this report, we determined the submitochondrial location of the MPI1 gene product and investigated whether it plays a direct role in the translocation of preproteins. By fractionation of mitochondria, the mature protein of 44 kDa was localized to the mitochondrial inner membrane and therefore termed MIM44. Import of the precursor of MIM44 required a membrane potential across the inner membrane and involved proteolytic processing of the precursor. A preprotein in transit across the mitochondrial membranes was cross-linked to MIM44, whereas preproteins arrested on the mitochondrial surface or fully imported proteins were not cross-linked. When preproteins were arrested at two distinct stages of translocation across the inner membrane, only preproteins at an early stage of translocation could be cross-linked to MIM44. Moreover, solubilized MIM44 was found to interact with in vitro-synthesized preproteins. We conclude that MIM44 is a component of the mitochondrial inner membrane import machinery and interacts with preproteins in an early step of translocation.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1609184113 ◽

2016 ◽

Vol 113 (38) ◽

pp. 10714-10719 ◽

Cited By ~ 28

Author(s):

Amélie A. Kelly ◽

Barbara Kalisch ◽

Georg Hölzl ◽

Sandra Schulze ◽

Juliane Thiele ◽

...

Keyword(s):

Fusion Proteins ◽

Chloroplast Envelope ◽

Envelope Membrane ◽

Lipid Transfer ◽

Terminal Sequence ◽

Outer Envelope ◽

Outer Envelope Membrane ◽

Envelope Reconstruction ◽

Envelope Membranes

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.

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Two components of the chloroplast protein import apparatus, IAP86 and IAP75, interact with the transit sequence during the recognition and translocation of precursor proteins at the outer envelope.

The Journal of Cell Biology ◽

10.1083/jcb.134.2.315 ◽

1996 ◽

Vol 134 (2) ◽

pp. 315-327 ◽

Cited By ~ 112

Author(s):

Y Ma ◽

A Kouranov ◽

S E LaSala ◽

D J Schnell

Keyword(s):

Outer Membrane ◽

Protein Import ◽

Small Subunit ◽

Chloroplast Envelope ◽

Stable Complex ◽

Nucleoside Triphosphate ◽

Cross Linking ◽

Outer Envelope ◽

Bisphosphate Carboxylase ◽

Precursor Proteins

The interactions of precursor proteins with components of the chloroplast envelope were investigated during the early stages of protein import using a chemical cross-linking strategy. In the absence of energy, two components of the outer envelope import machinery, IAP86 and IAP75, cross-linked to the transit sequence of the precursor to the small subunit of ribulose-1, 5-bisphosphate carboxylase (pS) in a precursor binding assay. In the presence of concentrations of ATP or GTP that support maximal precursor binding to the envelope, cross-linking to the transit sequence occurred predominantly with IAP75 and a previously unidentified 21-kD polypeptide of the inner membrane, indicating that the transit sequence had inserted across the outer membrane. Cross-linking of envelope components to sequences in the mature portion of a second precursor, preferredoxin, was detected in the presence of ATP or GTP, suggesting that sequences distant from the transit sequence were brought into the vicinity of the outer membrane under these conditions. IAP75 and a third import component, IAP34, were coimmunoprecipitated with IAP86 antibodies from solubilized envelope membranes, indicating that these three proteins form a stable complex in the outer membrane. On the basis of these observations, we propose that IAP86 and IAP75 act as components of a multisubunit complex to mediate energy-independent recognition of the transit sequence and subsequent nucleoside triphosphate-induced insertion of the transit sequence across the outer membrane.

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