Record Details

Mitochondrial Import Of tRNA: Mechanistic Studies Of Post- Receptor Translocation

EPrints@IICB

View Archive Info
 
 
Field Value
 
Title Mitochondrial Import Of tRNA:
Mechanistic Studies Of Post-
Receptor Translocation
 
Creator Koley, Sandip
 
Subject Molecular & Human Genetics
 
Description Mitochondrial Import Of tRNA: Mechanistic Studies Of Post Receptor Translocation
Import of tRNA from the cytosol into the mitochondria of the kinetoplastid protozoon
Leishmania is a multistep process consisting of the binding of tRNA to specific receptors, its
transfer to an intermediate factor and translocation through the membrane. A functional RNA
import complex (RIC) isolated from Leishmania mitochondria or reconstituted by cloned and
expressed protein subunits are being employed in our laboratory to determine molecular basis of
these steps. The RIC from mitochondria of the kinetoplastid protozoon Leishmania tropica
induces translocation of tRNAs across artificial or natural membranes. tRNA import consists of a
number of discrete steps beginning with the binding of the substrate to a receptor subunit RIC1 or
RIC8A, followed by its transfer to a third subunit RIC9. Subsequently the tRNA passes into the
vesicle interior presumably through a membrane embedded translocation channel, the
composition and properties of which are largely unknown. Although the receptor binding and
transfer steps have been characterized in terms of role of specific subunits, little is known about
the final translocation step.
Specifically the objective of my research was to reconstitute functional import pore
complex on lipid bilayers and determine the permeability of such membrane vesicles by RIC
channel under different biochemical and biophysical conditions with tRNA and other different
small molecules, to carry out structural and functional studies of translocation by site directed
mutagenesis of important subunit gene. These experiments provided detailed insights into the
interaction of RIC with mitochondrial and other membranes.
I have shown that subunits RIC6 and RiC9 polymerize on the membrane to form the
hexamer (RIC6)3-(RIC9)3 in presence of RIC4A formed a R3 complex. The resultant complex R3
induced translocation of tRNA when the pH of the medium was lowered to ~6. This process was
independent of ATP and sensitive to the protonophore m-chlorocarbonylcyanide phenylhydrazone
(CCCP), and to the K+ ionophore valinomycin, but resistant to k+/h+ exchanger nigericin,
indicating the requirement of a membrane potrential Δψm generated by transmembrane proton
gradient. Indeed R3 mediated tRNA translocation could be induced at neutral pH by K+ diffusion
potential of 60-90mV (negative inside). However, translocation was independent of tRNA
sequence, and small molecules such as ATP, oligonucleotides, labeled amino acids could be taken
up by R3 liposomes at pH6.0 in contrast to large molecules such as linearized plasmid DNA
which fails the internalization process and shows the size specificity of R3 complex. My results
indicated that the (RIC6)3-(RIC9)3 complex forms a voltage gated pore similar to mitochondrial
protein import channels. To understand the critical residues involves in proton sensing points
mutants of RIC6 subunits were generated in all the 6 cysteines and 6 histidine residues as some of
those residues are involved in proton sensing in a homologous protein rieske fe-s protein of RIC6.
I have found out the critical cysteine and histidines along the protein chain are responsible for
proton sensing. Atomic force microscopy has been used to find out the nature of the reconstituted
channel in vitro. Atomic Force Microscopy of R3 revealed particles with an asymmetric surface
groove of ~20 nm rim diameter and ~1 nm depth.
 
Date 2014
 
Type Thesis
NonPeerReviewed
 
Format application/pdf
 
Identifier http://www.eprints.iicb.res.in/2007/1/THESIS.pdf
Koley, Sandip (2014) Mitochondrial Import Of tRNA: Mechanistic Studies Of Post- Receptor Translocation. PhD thesis, UNIVERSITY OF CALCUTTA.
 
Relation http://www.eprints.iicb.res.in/2007/