Tracing Human Mitochondrial Complex I Assembly by Use of GFP-Tagged Subunits

Cindy E.J. Dieteren; Werner J.H. Koopman; Leo G.J. Nijtmans

doi:10.1016/S0076-6879(08)04407-8

Tracing Human Mitochondrial Complex I Assembly by Use of GFP-Tagged Subunits

Cindy E.J. Dieteren^*, Werner J.H. Koopman, Leo G.J. Nijtmans

^*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceeding › Chapter › Academic › peer-review

6 Citations (Scopus)

Abstract

Disturbances in the assembly of mitochondrial complex I (CI) are a frequent cause of mitochondrial disorders. Several lines of evidence hint at a semi-sequential assembly pathway, in which the 45 individual subunits that form the holoenzyme are pieced together by means of smaller intermediates. To understand this process, it is necessary to explain the exact order, the rate-limiting steps, and the dynamics of subunit incorporation. In this chapter, we describe an approach to regulate the expression levels of an AcGFP₁-tagged subunit (NDUFS3) in mammalian cells by means of a tetracycline-inducible promoter. This strategy allows the study of the dynamics of CI assembly intermediates in living cells on native gels. After establishing that the AcGFP₁ tag does not interfere with the activity and assembly of the enzyme, we show how this system can be used to trace the labeled subunit in an induction pulse-chase experiment or to study its accumulation in specific assembly intermediates after inhibition of mitochondrial translation.

Original language	English
Title of host publication	Mitochondrial Function, Part A
Subtitle of host publication	Mitochondrial Electron Transport Complexes and Reactive Oxygen Species
Editors	William Allison, Immo Scheffler
Publisher	Elsevier
Chapter	7
Pages	133-151
Number of pages	19
ISBN (Print)	9780080877761
DOIs	https://doi.org/10.1016/S0076-6879(08)04407-8
Publication status	Published - 2009
Externally published	Yes

Publication series

Name	Methods in Enzymology
Publisher	Elsevier
Number	A
Volume	456
ISSN (Print)	0076-6879

Access to Document

10.1016/S0076-6879(08)04407-8

6 Citations
1 Chapter

The Use of Fluorescence Correlation Spectroscopy to Probe Mitochondrial Mobility and Intramatrix Protein Diffusion
Willems, P. H. G. M., Swarts, H. G., Hink, M. A. & Koopman, W. J. H., 2009, Mitochondrial Function, Part A: Mitochondrial Electron Transport Complexes and Reactive Oxygen Species. Allison, W. & Scheffler, I. (eds.). A ed. Elsevier, p. 287-302 16 p. (Methods in Enzymology; vol. 456, no. A).
Research output: Chapter in Book/Report/Conference proceeding › Chapter › Academic › peer-review
8 Citations (Scopus)

Cite this

Dieteren, C. E. J., Koopman, W. J. H., & Nijtmans, L. G. J. (2009). Tracing Human Mitochondrial Complex I Assembly by Use of GFP-Tagged Subunits. In W. Allison, & I. Scheffler (Eds.), Mitochondrial Function, Part A: Mitochondrial Electron Transport Complexes and Reactive Oxygen Species (pp. 133-151). (Methods in Enzymology; Vol. 456, No. A). Elsevier. https://doi.org/10.1016/S0076-6879(08)04407-8

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abstract = "Disturbances in the assembly of mitochondrial complex I (CI) are a frequent cause of mitochondrial disorders. Several lines of evidence hint at a semi-sequential assembly pathway, in which the 45 individual subunits that form the holoenzyme are pieced together by means of smaller intermediates. To understand this process, it is necessary to explain the exact order, the rate-limiting steps, and the dynamics of subunit incorporation. In this chapter, we describe an approach to regulate the expression levels of an AcGFP1-tagged subunit (NDUFS3) in mammalian cells by means of a tetracycline-inducible promoter. This strategy allows the study of the dynamics of CI assembly intermediates in living cells on native gels. After establishing that the AcGFP1 tag does not interfere with the activity and assembly of the enzyme, we show how this system can be used to trace the labeled subunit in an induction pulse-chase experiment or to study its accumulation in specific assembly intermediates after inhibition of mitochondrial translation.",

author = "Dieteren, {Cindy E.J.} and Koopman, {Werner J.H.} and Nijtmans, {Leo G.J.}",

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Dieteren, CEJ, Koopman, WJH & Nijtmans, LGJ 2009, Tracing Human Mitochondrial Complex I Assembly by Use of GFP-Tagged Subunits. in W Allison & I Scheffler (eds), Mitochondrial Function, Part A: Mitochondrial Electron Transport Complexes and Reactive Oxygen Species. Methods in Enzymology, no. A, vol. 456, Elsevier, pp. 133-151. https://doi.org/10.1016/S0076-6879(08)04407-8

Tracing Human Mitochondrial Complex I Assembly by Use of GFP-Tagged Subunits. / Dieteren, Cindy E.J.; Koopman, Werner J.H.; Nijtmans, Leo G.J.
Mitochondrial Function, Part A: Mitochondrial Electron Transport Complexes and Reactive Oxygen Species. ed. / William Allison; Immo Scheffler. Elsevier, 2009. p. 133-151 (Methods in Enzymology; Vol. 456, No. A).

Research output: Chapter in Book/Report/Conference proceeding › Chapter › Academic › peer-review

TY - CHAP

T1 - Tracing Human Mitochondrial Complex I Assembly by Use of GFP-Tagged Subunits

AU - Dieteren, Cindy E.J.

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AU - Nijtmans, Leo G.J.

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AB - Disturbances in the assembly of mitochondrial complex I (CI) are a frequent cause of mitochondrial disorders. Several lines of evidence hint at a semi-sequential assembly pathway, in which the 45 individual subunits that form the holoenzyme are pieced together by means of smaller intermediates. To understand this process, it is necessary to explain the exact order, the rate-limiting steps, and the dynamics of subunit incorporation. In this chapter, we describe an approach to regulate the expression levels of an AcGFP1-tagged subunit (NDUFS3) in mammalian cells by means of a tetracycline-inducible promoter. This strategy allows the study of the dynamics of CI assembly intermediates in living cells on native gels. After establishing that the AcGFP1 tag does not interfere with the activity and assembly of the enzyme, we show how this system can be used to trace the labeled subunit in an induction pulse-chase experiment or to study its accumulation in specific assembly intermediates after inhibition of mitochondrial translation.

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DO - 10.1016/S0076-6879(08)04407-8

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C2 - 19348886

AN - SCOPUS:63449126208

SN - 9780080877761

T3 - Methods in Enzymology

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BT - Mitochondrial Function, Part A

A2 - Allison, William

A2 - Scheffler, Immo

PB - Elsevier

ER -

Tracing Human Mitochondrial Complex I Assembly by Use of GFP-Tagged Subunits

Abstract

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Research output

The Use of Fluorescence Correlation Spectroscopy to Probe Mitochondrial Mobility and Intramatrix Protein Diffusion

Cite this