| 1. |
- Botelho, Salomé Calado, et al.
(författare)
-
TIM23-mediated insertion of transmembrane alpha-helices into the mitochondrial inner membrane
- 2011
-
Ingår i: EMBO Journal. - : Wiley. - 0261-4189 .- 1460-2075. ; 30:6, s. 1003-1011
-
Tidskriftsartikel (refereegranskat)abstract
- While overall hydrophobicity is generally recognized as the main characteristic of transmembrane (TM) alpha-helices, the only membrane system for which there are detailed quantitative data on how different amino acids contribute to the overall efficiency of membrane insertion is the endoplasmic reticulum (ER) of eukaryotic cells. Here, we provide comparable data for TIM23-mediated membrane protein insertion into the inner mitochondrial membrane of yeast cells. We find that hydrophobicity and the location of polar and aromatic residues are strong determinants of membrane insertion. These results parallel what has been found previously for the ER. However, we see striking differences between the effects elicited by charged residues flanking the TM segments when comparing the mitochondrial inner membrane and the ER, pointing to an unanticipated difference between the two insertion systems.
|
|
| 2. |
- Calado Botelho, Salomé, et al.
(författare)
-
Complement activation is involved in the hepatic injury caused by high-dose exposure of mice to perfluorooctanoic acid
- 2015
-
Ingår i: Chemosphere. - : Elsevier BV. - 0045-6535 .- 1879-1298. ; 129:SI, s. 225-231
-
Tidskriftsartikel (refereegranskat)abstract
- High-dose exposure of mice to perfluorooctanoate (PFOA) induces both hepatotoxicity and immunotoxicity. Here, we characterized the effects of TO-day dietary treatment with PFOA (0.002-0.02%, w/w) on the liver and complement system of male C57BL/6 mice. At all four doses, this compound caused hepatomegaly and reduced the serum level of triglycerides (an indicator for activation of the peroxisome proliferator-activated receptor-alpha (PPAR alpha)). At the highest dose (0.02%, w/w), this hepatomegaly was associated with the hepatic injury, as reflected in increased activity of alanine aminotranferase (ALAT) in the serum, severe hepatocyte hypertrophy and hepatocellular necrosis. PFOA-induced hepatic injury was associated with in vivo activation of the complement system as indicated by (i) significant attenuation of the serum activities of both the classical and alternative pathways; (ii) a marked reduction in the serum level of the complement factor 0; and (iii) deposition of the complement factor C3 fragment (C3a) in the hepatic parenchyma. PFOA did not activate the alternative pathway of complement in vitro. At doses lower than 0.02%, PFOA induced hepatocyte hypertrophy without causing liver injury or activating complement. These results reveal substantial involvement of activation of complement in the pathogenesis of PFOA-induced hepatotoxicity.
|
|
| 3. |
- Calado Botelho, Salomé, et al.
(författare)
-
Dislocation by the m-AAA Protease Increases the Threshold Hydrophobicity for Retention of Transmembrane Helices in the Inner Membrane of Yeast Mitochondria
- 2013
-
Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 288:7, s. 4792-4798
-
Tidskriftsartikel (refereegranskat)abstract
- Sorting of mitochondrial inner membrane proteins is a complex process in which translocons and proteases function in a concerted way. Many inner membrane proteins insert into the membrane via the TIM23 translocon, and some are then further acted upon by the mitochondrial m-AAA protease, a molecular motor capable of dislocating proteins from the inner membrane. This raises the possibility that the threshold hydrophobicity for the retention of transmembrane segments in the inner membrane is different depending on whether they belong to membrane proteins that are m-AAA protease substrates or not. Here, using model transmembrane segments engineered into m-AAA protease-dependent proteins, we show that the threshold hydrophobicity for membrane retention measured in yeast cells in the absence of a functional m-AAA protease is markedly lower than that measured in its presence. Whether a given hydrophobic segment in a mitochondrial inner membrane protein will ultimately form a transmembrane helix may therefore depend on whether or not it will be exposed to the pulling force exerted by the m-AAA protease during biogenesis.
|
|
| 4. |
- Calado Botelho, Salomé, et al.
(författare)
-
The association of Brahma with the Balbiani ring 1 gene of Chironomus tentans studied by immunoelectron microscopy and chromatin immunoprecipitation
- 2008
-
Ingår i: Insect molecular biology (Print). - : Wiley. - 0962-1075 .- 1365-2583. ; 17:5, s. 505-13
-
Tidskriftsartikel (refereegranskat)abstract
- Many steps of gene expression take place during transcription, and important functional information can thus be obtained by determining the distribution of specific factors along a transcribed gene. The Balbiani ring (BR) genes of the dipteran Chironomus tentans constitute a unique system for mapping the association of specific factors along a eukaryotic gene using immuno-electron microscopy (immuno-EM). The chromatin immunoprecipitation (ChIP) technique has provided an alternative, more general method for studying the association of proteins with specific genomic sequences. The immuno-EM and the ChIP methods suffer from different limitations, and thus a combination of both is advantageous. We have established optimal conditions for ChIP on chromatin extracted from the salivary glands of C. tentans, and we have analyzed the association of the SWI/SNF chromatin remodelling factor Brahma (Brm) with the BR1 gene by combined immuno-EM and ChIP. We show that Brm is not restricted to the promoter of the BR1 gene but is also associated with sequences in the middle and distal portions of the gene, which suggests that Brm has additional roles apart from regulating transcription initiation.
|
|
| 5. |
- Calado Botelho, Salomé, 1984-
(författare)
-
Translocation of proteins into and across the bacterial and mitochondrial inner membranes
- 2012
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Translocons are dynamic protein complexes with the ability to respond to specific signals and to transport polypeptides between two distinct environments. The Sec-type translocons are examples of such machineries that can interconvert between a pore forming conformation that translocates proteins across the membrane, and a channel-like conformation that integrates proteins into the membrane by lateral opening.This thesis aims to identify the signals encoded in the amino acid sequence of the translocating polypeptides that trigger the translocon to release defined segments into the membrane. The selected systems are the SecYEG translocon and the TIM23 complex responsible for inserting proteins into the bacterial and the mitochondrial inner membrane, respectively.These two translocons have been challenged in vivo with designed polypeptide segments and their insertion efficiency into the membrane was measured. This allowed identification of the sequence requirements that govern SecYEG- and TIM23-mediated membrane integration. For these two systems, “biological” hydrophobicity scales have been determined, giving the contributions of each of the 20 amino acids to the overall free energy of insertion of a transmembrane segment into the membrane.A closer analysis of the mitochondrial system has made it possible to additionally investigate the process of membrane dislocation mediated by the m-AAA protease. The threshold hydrophobicity required for a transmembrane segment to remain in the mitochondrial inner membrane after TIM23-mediated integration depends on whether the segment will be further acted upon by the m-AAA protease.Finally, an experimental approach is presented to distinguish between different protein sorting pathways at the level of the TIM23 complex, i.e., conservative sorting vs. stop-transfer pathways. The results suggest a connection between the metabolic state of the cell and the import of proteins into the mitochondria.
|
|
| 6. |
- Park, Kwangjin, et al.
(författare)
-
Dissecting Stop Transfer versus Conservative Sorting Pathways for Mitochondrial Inner Membrane Proteins in Vivo
- 2012
-
Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 288:3, s. 1521-1532
-
Tidskriftsartikel (refereegranskat)abstract
- Mitochondrial inner membrane proteins that carry an N-terminal presequence are sorted by one of two pathways: stop-transfer or conservative sorting. However, the sorting pathway is known for only a small number of proteins, in part due to lack of robust experimental tools with which to study. Here we present an approach that facilitates determination of inner membrane protein sorting pathways in vivo by fusing a mitochondrial inner membrane protein to the C-terminal part of Mgm1p containing the rhomboid cleavage region. We validated the Mgm1 fusion approach using a set of proteins for which the sorting pathway is known, and determined sorting pathways of inner membrane proteins for which the sorting mode is previously uncharacterized. For Sdh4p, a multi-spanning membrane protein, our results suggest that both conservative sorting and stop-transfer mechanisms are required for insertion. Furthermore, the sorting process of Mgm1 fusion proteins was analyzed under different growth conditions and yeast mutant strains that were defective in the import motor or the m-AAA protease function. Our results show that the sorting of mitochondrial proteins carrying moderately hydrophobic transmembrane segments is sensitive to cellular conditions, implying that mitochondrial import and membrane sorting in the physiological environment may be dynamically tuned.
|
|
| 7. |
- Öjemalm, Karin, et al.
(författare)
-
Quantitative Analysis of SecYEG-Mediated Insertion of Transmembrane alpha-Helices into the Bacterial Inner Membrane
- 2013
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 425:15, s. 2813-2822
-
Tidskriftsartikel (refereegranskat)abstract
- Most integral membrane proteins, both in prokaryotic and eukaryotic cells, are co-translationally inserted into the membrane via Sec-type translocons: the SecYEG complex in prokaryotes and the Sec61 complex in eukaryotes. The contributions of individual amino acids to the overall free energy of membrane insertion of single transmembrane alpha-helices have been measured for Sec61-mediated insertion into the endoplasmic reticulum (ER) membrane (Nature 450:1026-1030) but have not been systematically determined for SecYEG-mediated insertion into the bacterial inner membrane. We now report such measurements, carried out in Escherichia coli. Overall, there is a good correlation between the results found for the mammalian ER and the E. coli inner membrane, but the hydrophobicity threshold for SecYEG-mediated insertion is distinctly lower than that for Sec61-mediated insertion.
|
|
| 8. |
- Österberg, Marie, et al.
(författare)
-
Charged flanking residues control the efficiency of membrane insertion of the first transmembrane segment in yeast mitochondrial Mgm1p
- 2011
-
Ingår i: FEBS Letters. - : Wiley. - 0014-5793 .- 1873-3468. ; 585:8, s. 1238-1242
-
Tidskriftsartikel (refereegranskat)abstract
- Mgm1p is a nuclearly encoded GTPase important for mitochondrial fusion. Long and short isoforms of the protein are generated in a unique alternative topogenesis process in which the most N-terminal of two hydrophobic segments in the protein is inserted into the inner mitochondrial membrane in about half of the molecules and translocated across the inner membrane in the other half. In the latter population, the second hydrophobic segment is cleaved by the inner membrane protease Pcp1p, generating the short isoform. Here, we show that charged residues in the regions flanking the first segment critically affect the ratio between the two isoforms, providing new insight into the importance of charged residues in the insertion of proteins into the mitochondrial inner membrane.
|
|
