spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online November 21, 2007
doi: 10.1242/10.1242/jcs.018929

Journal of Cell Science 120, 4099-4106 (2007)
Published by The Company of Biologists 2007
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zara, V.
Right arrow Articles by Papatheodorou, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zara, V.
Right arrow Articles by Papatheodorou, P.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

Biogenesis of yeast dicarboxylate carrier: the carrier signature facilitates translocation across the mitochondrial outer membrane

Vincenzo Zara1,*, Alessandra Ferramosca1, Loredana Capobianco1,2, Katrin M. Baltz3,{ddagger}, Olga Randel3, Joachim Rassow3, Ferdinando Palmieri2 and Panagiotis Papatheodorou3

1 Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Università del Salento, Via Provinciale Lecce-Monteroni, I-73100 Lecce, Italy
2 Dipartimento Farmaco-Biologico, Università di Bari, I-70125 Bari, Italy
3 Institut für Physiologische Chemie, Medizinische Fakultät der Ruhr-Universität Bochum, D-44780 Bochum, Germany


Figure 1
View larger version (20K):
[in this window]
[in a new window]

  		Fig. 1. Import of radiolabelled DIC into isolated mitochondria. (A) The DIC is a protein of 298 residues containing six putative membrane-spanning domains (grey segments). A carrier signature is found in two positions (CS1 and CS2). (B) Wild-type DIC and a mutant version lacking the carrier signatures (exchange of the corresponding residues with alanine, {Delta}CS1/2) were synthesized in reticulocyte lysate in the presence of 35S-labelled methionine and centrifuged at 2°C for 45 minutes at 100,000 g. The pellets (P) and samples of the supernatants (S) were analyzed by SDS-PAGE and fluorography to determine the distribution of the radiolabelled DIC or DIC{Delta}CS1/2, respectively. Four samples were tested in parallel to calculate the standard deviation. (C) Mitochondrial import of DIC and derivatives lacking carrier signature CS1, CS2 or both. The 35S-labelled proteins were incubated with isolated yeast mitochondria at 25°C for different times, as indicated. The mitochondria were subsequently treated with proteinase K, re-isolated and the proteins were separated by SDS-PAGE. A PhosphorImager was used for quantification; the highest value was set to 100% (control). (D) The radiolabelled proteins were imported into mitochondria for 5 minutes and analyzed as in C. Standard samples of the reticulocyte lysates were included to allow the calculation of the import efficiency (n=3). (E) Import of DIC and derivatives containing the amino acid exchanges P33A, D35A or K38A. The radiolabelled proteins were imported into isolated mitochondria for different times and subsequently analyzed as in C. Bars represent standard deviation.

 

Figure 2
View larger version (10K):
[in this window]
[in a new window]

  		Fig. 2. Protein import into mitochondria lacking Tom70. (A) Import of Su9-DHFR. 35S-labelled Su9-DHFR (comprising the first 69 residues of Neurospora crassa ATP synthase subunit 9 fused to mouse dihydrofolate reductase) was imported into yeast mitochondria isolated from a strain lacking the import receptor Tom70 ({Delta}tom70) or from the corresponding wild-type strain (WT). For calculation of the import efficiency, the total amount of the radiolabelled preprotein in the samples was set to 100%. (B) Import of wild-type DIC into mitochondria from the same preparation as in A. (C) Import of DIC{Delta}CS1/2 into mitochondria from the same preparation as in A. Bars represent standard deviation.

 

Figure 3
View larger version (27K):
[in this window]
[in a new window]

  		Fig. 3. Accessibility of DIC to proteases after import into mitochondria. 35S-labelled wild-type DIC and the mutant versions {Delta}CS1, {Delta}CS2 and {Delta}CS1/2 were synthesized in reticulocyte lysate and incubated with isolated yeast mitochondria for 5 minutes at 25°C. The mitochondria were then cooled to 0°C and re-isolated by centrifugation. The mitochondria were then resuspended either in 1 mM EDTA, 10 mM MOPS/KOH, pH 7.2 to allow swelling (+SW) of the mitochondria and rupture of the outer membrane, or in 250 mM sucrose, 1 mM EDTA, 10 mM MOPS/KOH, pH 7.2 to keep the mitochondria intact (–SW). All samples were treated with 250 µg/ml proteinase K for 20 minutes at 0°C. Mitochondria and mitoplasts were re-isolated, the proteins were separated by SDS-PAGE, blotted on nitrocellulose and the radiolabelled proteins were visualized using a PhosphorImager (upper panel). For quantification of each construct, the amount of 35S-labelled protein in the –SW sample was set to 100%. The nitrocellulose was eventually immuno-decorated with polyclonal antibodies directed against the hydrophilic N-terminus of the inner-membrane protein Tim23. PK, proteinase K. Bars represent standard deviation.

 

Figure 4
View larger version (49K):
[in this window]
[in a new window]

  		Fig. 4. Maturation of DIC in the mitochondrial inner membrane. (A) Analysis by BN-PAGE. 35S-labelled DIC and the derivatives {Delta}CS1, {Delta}CS2 and {Delta}CS1/2 were imported into isolated yeast mitochondria for 20 minutes at 25°C, the mitochondria were treated with proteinase K, re-isolated, dissolved in the presence of 1% digitonin, and separated by BN-PAGE. The radiolabelled proteins were visualized using a PhosphorImager. Some samples contained valinomycin to dissipate the mitochondrial membrane potential (–{Delta}{Psi}). As indicated, mature carrier proteins (import stage V) were usually detected by this method at a range corresponding to 80-120 kDa. (B) 2D analysis of DIC{Delta}CS1. A stripe from a BN-PAGE as shown in A was excised and layered on top of an SDS-PAGE for separation of the proteins in a second dimension. The radiolabelled proteins were subsequently visualized using a PhosphorImager. Upper panel, stripe from a DIC{Delta}CS1 BN-PAGE sample run in parallel; lower panel, SDS-PAGE. (C) Affinity of DIC for hydroxyapatite. Radiolabelled DIC and DIC{Delta}CS1/2 were imported into mitochondria for 15 minutes at 25°C. As indicated, parallel samples contained valinomycin to dissipate the membrane potential (–{Delta}{Psi}). The mitochondria were then treated with proteinase K, re-isolated and solubilized in the presence of 2.5% Triton X-100. Following a clarifying spin for 5 minutes at 16,000 g, the supernatants were passed through small columns containing hydroxyapatite. The proteins of the eluates were collected by precipitation with trichloro-acetic acid and analyzed by SDS-PAGE and fluorography.

 

Figure 5
View larger version (18K):
[in this window]
[in a new window]

  		Fig. 5. Transport activity of DIC after reconstitution in liposomes. The sarkosyl-solubilized proteins were reconstituted into liposomes in the presence of 20 mM potassium phosphate, pH 7.2. The external substrate was removed from proteoliposomes on Sephadex G-75 columns. The transport was started by adding 0.1 mM [33P]-phosphate and terminated after 10 minutes. Finally, the external substrate was removed on Sephadex G-75 columns and the accumulation of labelled substrate in the proteoliposomes was measured. The values reported in the figure represent the means ± s.d. (n=4). WT, wild type.

 

Figure 6
View larger version (20K):
[in this window]
[in a new window]

  		Fig. 6. Stages of DIC import into mitochondria. (I) Newly synthesized DIC is bound to chaperone proteins in the cytosol; (II) DIC then becomes bound to the import receptor Tom70; (III*) DIC is bound the Tim9-Tim10 complex in the intermembrane space [as defined by Zara et al. (Zara et al., 2001Go)]; (IV) insertion into the inner-membrane Tim22 complex; V, mature DIC. The carrier signature facilitates the transition from stage II to stage III* and is essential in the transport activity of the mature protein (large arrows). IM, inner membrane; IMS, intermembrane space; OM, outer membrane; R, receptor.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2007