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Translocation of proteins into and across the bacterial and mitochondrial inner membranes
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. (Gunnar von Heijne)
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

 

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.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2012. , 86 p.
Keyword [en]
Escherichia coli, mitochondria, Saccharomyces cerevisiae, SecYEG, TIM23, transmembrane helix
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-83234ISBN: 978-91-7447-600-2 (print)OAI: oai:DiVA.org:su-83234DiVA: diva2:575366
Public defence
2013-01-11, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of doctoral defence the following papers were unpublished and had a status as follows: Paper nr. 1: Manuscript; Paper nr. 4: Manuscript

Available from: 2012-12-20 Created: 2012-12-06 Last updated: 2012-12-21Bibliographically approved
List of papers
1. Quantitative analysis of SecYEG-mediated insertion of transmembrane α-helices into the bacterial inner membrane
Open this publication in new window or tab >>Quantitative analysis of SecYEG-mediated insertion of transmembrane α-helices into the bacterial inner membrane
2012 (English)Manuscript (preprint) (Other academic)
Abstract [en]

Most integral membrane proteins, both inprokaryotic and eukaryotic cells, are cotranslationallyinserted into the membrane viaSec-type translocons: the SecYEG complex inprokaryotes and the Sec61 complex ineukaryotes. The contributions of individualamino acids to the overall free energy ofmembrane insertion of single transmembraneα-helices have been measured for Sec61-mediated insertion into the endoplasmicreticular (ER) membrane (Nature 450:1026-1030), but have not been systematicallydetermined for SecYEG-mediated insertioninto the bacterial inner membrane. We nowreport such measurements, carried out inEscherichia coli. Overall, there is a goodcorrelation between the results found for themammalian ER and the E. coli innermembrane, but the hydrophobicity thresholdfor SecYEG-mediated insertion is distinctlylower than for Sec61-mediated insertion.

National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-83482 (URN)
Available from: 2012-12-11 Created: 2012-12-11 Last updated: 2017-11-16Bibliographically approved
2. TIM23-mediated insertion of transmembrane alpha-helices into the mitochondrial inner membrane
Open this publication in new window or tab >>TIM23-mediated insertion of transmembrane alpha-helices into the mitochondrial inner membrane
Show others...
2011 (English)In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 30, no 6, 1003-1011 p.Article in journal (Refereed) Published
Abstract [en]

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.

Keyword
CoxVa, membrane protein, Mgm1p, mitochondria, TIM23
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-69461 (URN)10.1038/emboj.2011.29 (DOI)000289672900006 ()
Note
authorCount :8Available from: 2012-01-13 Created: 2012-01-12 Last updated: 2017-12-08Bibliographically approved
3. Charged flanking residues control the efficiency of membrane insertion of the first transmembrane segment in yeast mitochondrial Mgm1p
Open this publication in new window or tab >>Charged flanking residues control the efficiency of membrane insertion of the first transmembrane segment in yeast mitochondrial Mgm1p
2011 (English)In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 585, no 8, 1238-1242 p.Article in journal (Refereed) Published
Abstract [en]

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.

Keyword
Mgm1p, Mitochondria, TIM23, Transmembrane helix, Yeast, Saccharomyces cerevisiae
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-68816 (URN)10.1016/j.febslet.2011.03.056 (DOI)000289505400021 ()
Note
authorCount :4Available from: 2012-01-09 Created: 2012-01-07 Last updated: 2017-12-08Bibliographically approved
4. Dislocation by the m-AAA protease increases the threshold hydrophobicity for retention of transmembrane helices in the inner membrane of yeast mitochondria
Open this publication in new window or tab >>Dislocation by the m-AAA protease increases the threshold hydrophobicity for retention of transmembrane helices in the inner membrane of yeast mitochondria
2012 (English)Manuscript (preprint) (Other academic)
Abstract [en]

Sorting of mitochondrial inner membraneproteins is a complex process where transloconsand proteases function in a concerted way.Many inner membrane proteins insert into themembrane via the TIM23 translocon and someare then further acted upon by themitochondrial m-AAA protease, a molecularmotor capable of dislocating proteins from theinner membrane. This raises the possibility thatthe threshold hydrophobicity for the retentionof transmembrane segments in the innermembrane is different, depending on whetherthey belong to membrane proteins that are m-AAA protease substrate or not. Here, usingmodel transmembrane segments engineeredinto m-AAA protease-dependent proteins, weshow that the threshold hydrophobicity formembrane retention measured in yeast cells inthe absence of a functional m-AAA protease ismarkedly lower than that measured in itspresence. Whether a given hydrophobicsegment in a mitochondrial inner membraneprotein will ultimately form a transmembranehelix may therefore depend on whether or not,during biogenesis, it will be exposed to thepulling force exerted by the m-AAA protease.

National Category
Natural Sciences
Identifiers
urn:nbn:se:su:diva-83481 (URN)
Available from: 2012-12-11 Created: 2012-12-11 Last updated: 2012-12-12Bibliographically approved
5. Dissecting Stop Transfer versus Conservative Sorting Pathways for Mitochondrial Inner Membrane Proteins in Vivo
Open this publication in new window or tab >>Dissecting Stop Transfer versus Conservative Sorting Pathways for Mitochondrial Inner Membrane Proteins in Vivo
Show others...
2012 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, no 3, 1521-1532 p.Article in journal (Refereed) Published
Abstract [en]

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.

National Category
Biochemistry and Molecular Biology Biophysics
Identifiers
urn:nbn:se:su:diva-83479 (URN)10.1074/jbc.M112.409748 (DOI)000313751400010 ()
Available from: 2012-12-11 Created: 2012-12-11 Last updated: 2017-12-07Bibliographically approved

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