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Sequence and structural requirements of a mitochondrial protein import signal defined by saturation cassette mutagenesis

Bedwell, David M. and Strobel, Scott A. and Yun, Kyuson and Jongeward, Gregg D. and Emr, Scott D. (1989) Sequence and structural requirements of a mitochondrial protein import signal defined by saturation cassette mutagenesis. Molecular and Cellular Biology, 9 (3). pp. 1014-1025. ISSN 0270-7306. PMCID PMC362691.

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The Saccharomyces cerevisiae F1-ATPase beta subunit precursor contains redundant mitochondrial protein import information at its NH2 terminus (D. M. Bedwell, D. J. Klionsky, and S. D. Emr, Mol. Cell. Biol. 7:4038-4047, 1987). To define the critical sequence and structural features contained within this topogenic signal, one of the redundant regions (representing a minimal targeting sequence) was subjected to saturation cassette mutagenesis. Each of 97 different mutant oligonucleotide isolates containing single (32 isolates), double (45 isolates), or triple (20 isolates) point mutations was inserted in front of a beta-subunit gene lacking the coding sequence for its normal import signal (codons 1 through 34 were deleted). The phenotypic and biochemical consequences of these mutations were then evaluated in a yeast strain deleted for its normal beta-subunit gene (delta atp2). Consistent with the lack of an obvious consensus sequence for mitochondrial protein import signals, many mutations occurring throughout the minimal targeting sequence did not significantly affect its import competence. However, some mutations did result in severe import defects. In these mutants, beta-subunit precursor accumulated in the cytoplasm, and the yeast cells exhibited a respiration defective phenotype. Although point mutations have previously been identified that block mitochondrial protein import in vitro, a subset of the mutations reported here represents the first single missense mutations that have been demonstrated to significantly block mitochondrial protein import in vivo. The previous lack of such mutations in the beta-subunit precursor apparently relates to the presence of redundant import information in this import signal. Together, our mutants define a set of constraints that appear to be critical for normal activity of this (and possibly other) import signals. These include the following: (i) mutant signals that exhibit a hydrophobic moment greater than 5.5 for the predicted amphiphilic alpha-helical conformation of this sequence direct near normal levels of beta-subunit import (ii) at least two basic residues are necessary for efficient signal function, (iii) acidic amino acids actively interfere with import competence, and (iv) helix-destabilizing residues also interfere with signal function. These experimental observations provide support for mitochondrial protein import models in which both the structure and charge of the import signal play a critical role in directing mitochondrial protein targeting and import.

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Additional Information:© 1989, American Society for Microbiology Received 20 October 1988/Accepted 9 December 1988 We thank Suzanna Horvath for advice concerning the design of the mixed oligonucleotide, Jeff Schatz and Mike Douglas for providing antisera and monoclonal antibodies, and Keith Rosema for writing the computer program used to calculate the hydrophobic moment values. We also thank Bruce Horazdovsky for critically reading the manuscript. This study was supported by Public Health Service grant GM-32703 from the National Institues of Health. S.D.E. is a Presidential Young Investigator supported by National Science Foundation grant DCB-8451633. D.M.B. was supported by a Research Fellowship from the American Cancer Society. S.A.S. and G.D.J. were supported by a predoctoral training grant from the National Institutes of Health.
Issue or Number:3
PubMed Central ID:PMC362691
Record Number:CaltechAUTHORS:BEDmcb89
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:2181
Deposited By: Archive Administrator
Deposited On:13 Mar 2006
Last Modified:02 Oct 2019 22:50

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