Molecular and Cellular Biology, October 2000, p. 7410-7417, Vol. 20, No. 19
Genetic Engineering Laboratory, Indian
Institute of Chemical Biology, Calcutta 700032, India
Received 29 February 2000/Returned for modification 28 March
2000/Accepted 20 June 2000
Nucleus-encoded tRNAs are selectively imported into the
mitochondrion of Leishmania, a kinetoplastid protozoan. An
oligoribonucleotide constituting the D stem-loop import signal of
tRNATyr(GUA) was efficiently transported into the
mitochondrial matrix in organello as well as in vivo. Transfer through
the inner membrane could be uncoupled from that through the outer
membrane and was resistant to antibody against the outer membrane
receptor TAB. A number of mutations in the import signal had
differential effects on outer and inner membrane transfer. Some mutants
which efficiently traversed the outer membrane were unable to enter the
matrix. Conversely, restoration of the loop-closing GC pair in reverse resulted in reversion of transfer through the inner, but not the outer,
membrane, and binding of the RNA to the inner membrane was restored.
These experiments indicate the presence at the two membranes of
receptors with distinct specificities which mediate stepwise transfer
into the mitochondrial matrix. The combination of oligonucleotide
mutagenesis and biochemical fractionation may provide a general tool
for the identification of tRNA transport factors.
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Mutations in a tRNA Import Signal Define Distinct
Receptors at the Two Membranes of Leishmania
Mitochondria
*
Corresponding author. Mailing address: Genetic
Engineering Laboratory, Indian Institute of Chemical Biology, 4 Raja
S. C. Mullick Rd., Calcutta 700032, India. Phone: 91 33 473 3491, ext. 136. Fax: 91 33 473 5197/0284. E-mail:
IICHBIO{at}GIASCL01.VSNL.NET.IN.
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