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Molecular and Cellular Biology, October 2000, p. 7410-7417, Vol. 20, No. 19
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

Subhendra Nath Bhattacharyya, Shankar Mukherjee, and Samit Adhya*

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.

* 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.

Molecular and Cellular Biology, October 2000, p. 7410-7417, Vol. 20, No. 19
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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