Probing the environment along the protein import pathways in yeast mitochondria by site-specific photocrosslinking

Takashi Kanamori, Shuh Ichi Nishikawa, Injae Shin, Peter G. Schultz, Toshiya Endo

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Abstract

Artificially aminoacylated suppressor tRNAs were used to introduce photoreactive amino acids into model mitochondrial precursor proteins to probe the environment along the protein import pathway. Amino acids with benzophenone side chains of various lengths [DL-2-amino-3-(p- benzoylphenyl)propanoic acid (1) and DL-2-amino-5-(p-benzoylphenyl)pentanoic acid (2)] were incorporated at specific sites throughout the cytochrome b2- dihydrofolate reductase fusion proteins, pb2(220)-DHFR and pb2Δ19(220)- DHFR, which were destined for the intermembrane space and the matrix in mitochondria, respectively. In vitro import of pb2(220)-DHFR and pb2Δ19(220)-DHFR bearing 1 or 2 into isolated yeast mitochondria was arrested so that the N terminus reached the intermembrane space or the matrix, respectively, while the DHFR domain remained at the mitochondrial surface. The matrix-targeted pb2Δ19(220)-DHFR was photocrosslinked to Tom40 in the outer membrane, Tim44 in the inner membrane, and Ssc1p in the matrix, suggesting that the protein has an extended conformation in the import channels. On the other hand, incorporation of 2 at various positions in the 50-residue segment of intermembrane-space-targeted pb2(220)-DHFR gave photocrosslinks only to Tom40, suggesting that the segment is not in an extended conformation, but localized near Tom40. The N-terminal portion of pb2(220)-DHFR, but not pb2Δ19(220)-DHFR, was photocrosslinked to an as- yet-unidentified mitochondrial component to generate a 95-kDa crosslinked product.

Original languageEnglish
Pages (from-to)485-490
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume94
Issue number2
DOIs
Publication statusPublished - 1997 Jan 21

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Mitochondria
Yeasts
L-Lactate Dehydrogenase (Cytochrome)
Pentanoic Acids
Amino Acids
Tetrahydrofolate Dehydrogenase
Proteins
Membranes
Mitochondrial Proteins
Transfer RNA
propionic acid
In Vitro Techniques
benzophenone
3,2'-difucosyllactose

All Science Journal Classification (ASJC) codes

  • General

Cite this

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title = "Probing the environment along the protein import pathways in yeast mitochondria by site-specific photocrosslinking",
abstract = "Artificially aminoacylated suppressor tRNAs were used to introduce photoreactive amino acids into model mitochondrial precursor proteins to probe the environment along the protein import pathway. Amino acids with benzophenone side chains of various lengths [DL-2-amino-3-(p- benzoylphenyl)propanoic acid (1) and DL-2-amino-5-(p-benzoylphenyl)pentanoic acid (2)] were incorporated at specific sites throughout the cytochrome b2- dihydrofolate reductase fusion proteins, pb2(220)-DHFR and pb2Δ19(220)- DHFR, which were destined for the intermembrane space and the matrix in mitochondria, respectively. In vitro import of pb2(220)-DHFR and pb2Δ19(220)-DHFR bearing 1 or 2 into isolated yeast mitochondria was arrested so that the N terminus reached the intermembrane space or the matrix, respectively, while the DHFR domain remained at the mitochondrial surface. The matrix-targeted pb2Δ19(220)-DHFR was photocrosslinked to Tom40 in the outer membrane, Tim44 in the inner membrane, and Ssc1p in the matrix, suggesting that the protein has an extended conformation in the import channels. On the other hand, incorporation of 2 at various positions in the 50-residue segment of intermembrane-space-targeted pb2(220)-DHFR gave photocrosslinks only to Tom40, suggesting that the segment is not in an extended conformation, but localized near Tom40. The N-terminal portion of pb2(220)-DHFR, but not pb2Δ19(220)-DHFR, was photocrosslinked to an as- yet-unidentified mitochondrial component to generate a 95-kDa crosslinked product.",
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Probing the environment along the protein import pathways in yeast mitochondria by site-specific photocrosslinking. / Kanamori, Takashi; Nishikawa, Shuh Ichi; Shin, Injae; Schultz, Peter G.; Endo, Toshiya.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 94, No. 2, 21.01.1997, p. 485-490.

Research output: Contribution to journalArticle

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T1 - Probing the environment along the protein import pathways in yeast mitochondria by site-specific photocrosslinking

AU - Kanamori, Takashi

AU - Nishikawa, Shuh Ichi

AU - Shin, Injae

AU - Schultz, Peter G.

AU - Endo, Toshiya

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