scholarly journals Tic20 and Tic22 Are New Components of the Protein Import Apparatus at the Chloroplast Inner Envelope Membrane

1998 ◽  
Vol 143 (4) ◽  
pp. 991-1002 ◽  
Author(s):  
Andrei Kouranov ◽  
Xuejun Chen ◽  
Bruno Fuks ◽  
Danny J. Schnell
Keyword(s):  
Protein Import ◽  
Intermediate Stage ◽  
Chloroplast Envelope ◽  
Intermembrane Space ◽  
Outer Face ◽  
Envelope Membrane ◽  
Stromal Compartment ◽  
Inner Membrane ◽  
General Protein ◽  
Inner Envelope

Two components of the chloroplast envelope, Tic20 and Tic22, were previously identified as candidates for components of the general protein import machinery by their ability to covalently cross-link to nuclear-encoded preproteins trapped at an intermediate stage in import across the envelope (Kouranov, A., and D.J. Schnell. 1997. J. Cell Biol. 139:1677–1685). We have determined the primary structures of Tic20 and Tic22 and investigated their localization and association within the chloroplast envelope. Tic20 is a 20-kD integral membrane component of the inner envelope membrane. In contrast, Tic22 is a 22-kD protein that is located in the intermembrane space between the outer and inner envelope membranes and is peripherally associated with the outer face of the inner membrane. Tic20, Tic22, and a third inner membrane import component, Tic110, associate with import components of the outer envelope membrane. Preprotein import intermediates quantitatively associate with this outer/inner membrane supercomplex, providing evidence that the complex corresponds to envelope contact sites that mediate direct transport of preproteins from the cytoplasm to the stromal compartment. On the basis of these results, we propose that Tic20 and Tic22 are core components of the protein translocon of the inner envelope membrane of chloroplasts.

scholarly journals Tim23–Tim50 pair coordinates functions of translocators and motor proteins in mitochondrial protein import

2009 ◽  
Vol 184 (1) ◽  
pp. 129-141 ◽  
Author(s):  
Yasushi Tamura ◽  
Yoshihiro Harada ◽  
Takuya Shiota ◽  
Koji Yamano ◽  
Kazuaki Watanabe ◽  
...  
Keyword(s):  
Motor Proteins ◽  
Protein Import ◽  
Protein Translocation ◽  
Mitochondrial Protein ◽  
Intermembrane Space ◽  
Mitochondrial Protein Import ◽  
Inner Membrane ◽  
The Matrix ◽  
General Protein ◽  
Mitochondrial Hsp70

Mitochondrial protein traffic requires coordinated operation of protein translocator complexes in the mitochondrial membrane. The TIM23 complex translocates and inserts proteins into the mitochondrial inner membrane. Here we analyze the intermembrane space (IMS) domains of Tim23 and Tim50, which are essential subunits of the TIM23 complex, in these functions. We find that interactions of Tim23 and Tim50 in the IMS facilitate transfer of precursor proteins from the TOM40 complex, a general protein translocator in the outer membrane, to the TIM23 complex. Tim23–Tim50 interactions also facilitate a late step of protein translocation across the inner membrane by promoting motor functions of mitochondrial Hsp70 in the matrix. Therefore, the Tim23–Tim50 pair coordinates the actions of the TOM40 and TIM23 complexes together with motor proteins for mitochondrial protein import.

scholarly journals Reconstitution of protein targeting to the inner envelope membrane of chloroplasts

2006 ◽  
Vol 175 (2) ◽  
pp. 249-259 ◽  
Author(s):  
Ming Li ◽  
Danny J. Schnell
Keyword(s):  
Lipid Metabolism ◽  
Protein Targeting ◽  
Chloroplast Envelope ◽  
Envelope Membrane ◽  
Stromal Compartment ◽  
Intermediate Size ◽  
Envelope Membranes ◽  
Inner Envelope

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.

scholarly journals A Nuclear-coded Chloroplastic Inner Envelope Membrane Protein Uses a Soluble Sorting Intermediate upon Import into the Organelle

1997 ◽  
Vol 137 (6) ◽  
pp. 1279-1286 ◽  
Author(s):  
Jens Lübeck ◽  
Lisa Heins ◽  
Jürgen Soll
Keyword(s):  
Protein Import ◽  
Proximal Part ◽  
Small Subunit ◽  
Intermembrane Space ◽  
Envelope Membrane ◽  
Bisphosphate Carboxylase ◽  
Hybrid Proteins ◽  
Chloroplast Stroma ◽  
Inner Envelope

The chloroplastic inner envelope protein of 110 kD (IEP110) is part of the protein import machinery in the pea. Different hybrid proteins were constructed to assess the import and sorting pathway of IEP110. The IEP110 precursor (pIEP110) uses the general import pathway into chloroplasts, as shown by the mutual exchange of presequences with the precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase (pSSU). Sorting information to the chloroplastic inner envelope is contained in an NH2-proximal part of mature IEP110 (110N). The NH2-terminus serves to anchor the protein into the membrane. Large COOH-terminal portions of this protein (80–90 kD) are exposed to the intermembrane space in situ. Successful sorting and integration of IEP110 and the derived constructs into the inner envelope are demonstrated by the inaccessability of processed mature protein to the protease thermolysin but accessibility to trypsin, i.e., the imported protein is exposed to the intermembrane space. A hybrid protein consisting of the transit sequence of SSU, the NH2-proximal part of mature IEP110, and mature SSU (tpSSU-110N-mSSU) is completely imported into the chloroplast stroma, from which it can be recovered as soluble, terminally processed 110NmSSU. The soluble 110N-mSSU then enters a reexport pathway, which results not only in the insertion of 110N-mSSU into the inner envelope membrane, but also in the extrusion of large portions of the protein into the intermembrane space. We conclude that chloroplasts possess a protein reexport machinery for IEPs in which soluble stromal components interact with a membrane-localized translocation machinery.

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

1996 ◽  
Vol 132 (1) ◽  
pp. 63-75 ◽  
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.

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

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.

Mechanism of protein import across the chloroplast envelope

2000 ◽  
Vol 28 (4) ◽  
pp. 485-491 ◽  
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.

scholarly journals Mutant huntingtin disrupts mitochondrial proteostasis by interacting with TIM23

2019 ◽  
Vol 116 (33) ◽  
pp. 16593-16602 ◽  
Author(s):  
Svitlana Yablonska ◽  
Vinitha Ganesan ◽  
Lisa M. Ferrando ◽  
JinHo Kim ◽  
Anna Pyzel ◽  
...  
Keyword(s):  
Cell Lines ◽  
Protein Import ◽  
Mitochondrial Protein ◽  
Biological Significance ◽  
Intermembrane Space ◽  
Mutant Huntingtin ◽  
Mitochondrial Protein Import ◽  
Inner Membrane ◽  
Mitochondrial Proteome ◽  
Brain Tissues

Mutant huntingtin (mHTT), the causative protein in Huntington’s disease (HD), associates with the translocase of mitochondrial inner membrane 23 (TIM23) complex, resulting in inhibition of synaptic mitochondrial protein import first detected in presymptomatic HD mice. The early timing of this event suggests that it is a relevant and direct pathophysiologic consequence of mHTT expression. We show that, of the 4 TIM23 complex proteins, mHTT specifically binds to the TIM23 subunit and that full-length wild-type huntingtin (wtHTT) and mHTT reside in the mitochondrial intermembrane space. We investigated differences in mitochondrial proteome between wtHTT and mHTT cells and found numerous proteomic disparities between mHTT and wtHTT mitochondria. We validated these data by quantitative immunoblotting in striatal cell lines and human HD brain tissue. The level of soluble matrix mitochondrial proteins imported through the TIM23 complex is lower in mHTT-expressing cell lines and brain tissues of HD patients compared with controls. In mHTT-expressing cell lines, membrane-bound TIM23-imported proteins have lower intramitochondrial levels, whereas inner membrane multispan proteins that are imported via the TIM22 pathway and proteins integrated into the outer membrane generally remain unchanged. In summary, we show that, in mitochondria, huntingtin is located in the intermembrane space, that mHTT binds with high-affinity to TIM23, and that mitochondria from mHTT-expressing cells and brain tissues of HD patients have reduced levels of nuclearly encoded proteins imported through TIM23. These data demonstrate the mechanism and biological significance of mHTT-mediated inhibition of mitochondrial protein import, a mechanism likely broadly relevant to other neurodegenerative diseases.

scholarly journals The biogenesis of mitochondrial intermembrane space proteins

2020 ◽  
Vol 401 (6-7) ◽  
pp. 737-747 ◽  
Author(s):  
Ruairidh Edwards ◽  
Sarah Gerlich ◽  
Kostas Tokatlidis
Keyword(s):  
Protein Import ◽  
Cellular Stress ◽  
Stress Conditions ◽  
Intermembrane Space ◽  
Dynamic Changes ◽  
Inner Membrane ◽  
Integrated Network ◽  
Response To Stress ◽  
Mitochondrial Intermembrane Space

AbstractThe mitochondrial intermembrane space (IMS) houses a large spectrum of proteins with distinct and critical functions. Protein import into this mitochondrial sub-compartment is underpinned by an intriguing variety of pathways, many of which are still poorly understood. The constricted volume of the IMS and the topological segregation by the inner membrane cristae into a bulk area surrounded by the boundary inner membrane and the lumen within the cristae is an important factor that adds to the complexity of the protein import, folding and assembly processes. We discuss the main import pathways into the IMS, but also how IMS proteins are degraded or even retro-translocated to the cytosol in an integrated network of interactions that is necessary to maintain a healthy balance of IMS proteins under physiological and cellular stress conditions. We conclude this review by highlighting new and exciting perspectives in this area with a view to develop a better understanding of yet unknown, likely unconventional import pathways, how presequence-less proteins can be targeted and the basis for dual localisation in the IMS and the cytosol. Such knowledge is critical to understanding the dynamic changes of the IMS proteome in response to stress, and particularly important for maintaining optimal mitochondrial fitness.

scholarly journals Role of the intermembrane-space domain of the preprotein receptor Tom22 in protein import into mitochondria.

1996 ◽  
Vol 16 (8) ◽  
pp. 4035-4042 ◽  
Author(s):  
D A Court ◽  
F E Nargang ◽  
H Steiner ◽  
R S Hodges ◽  
W Neupert ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Protein Import ◽  
Protein Translocation ◽  
Specific Binding ◽  
Mitochondrial Outer Membrane ◽  
Intermembrane Space ◽  
Inner Membrane ◽  
Terminal Domain ◽  
Tom Complex

Tom22 is an essential component of the protein translocation complex (Tom complex) of the mitochondrial outer membrane. The N-terminal domain of Tom22 functions as a preprotein receptor in cooperation with Tom20. The role of the C-terminal domain of Tom22, which is exposed to the intermembrane space (IMS), in its own assembly into the Tom complex and in the import of other preproteins was investigated. The C-terminal domain of Tom22 is not essential for the targeting and assembly of this protein, as constructs lacking part or all of the IMS domain became imported into mitochondria and assembled into the Tom complex. Mutant strains of Neurospora expressing the truncated Tom22 proteins were generated by a novel procedure. These mutants displayed wild-type growth rates, in contrast to cells lacking Tom22, which are not viable. The import of proteins into the outer membrane and the IMS of isolated mutant mitochondria was not affected. Some but not all preproteins destined for the matrix and inner membrane were imported less efficiently. The reduced import was not due to impaired interaction of presequences with their specific binding site on the trans side of the outer membrane. Rather, the IMS domain of Tom22 appears to slightly enhance the efficiency of the transfer of these preproteins to the import machinery of the inner membrane.

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