PROTEIN IMPORT INTO MITOCHONDRIA

Project Details

Description

The goal of this proposal is to define the mechanism by which GTP
participates in mitochondrial protein import using yeast as a model
system. We have demonstrated for first time that GTP hydrolysis
plays a direct and essential role during import of urea-denatured
precursors into the matrix. Import of these denatured precursors
does not depend on ATP-dependent interactions with cytosolic
chaperones. Both external GTP and matrix ATP are required for
efficient import. Protein import into or across mitochondrial outer
and inner membranes is a multi-step process. GTP may regulate
recognition and subsequent unfolding of precursors at the
mitochondrial surface and/or provide energy for transmembrane
movement of precursors into the organelle. The first objective is to
define the exact steps that require GTP. The GTP effect is likely
mediated by GTP-binding proteins. Thus far, a GTP-binding protein
has not been identified as part of the outer (Tom) or inner membrane
(Tim) translocases. The second objective is to identify these
critical GTP-binding proteins. A chimeric protein (pPHPrA) has been
constructed, comprising an authentic N-terminal mitochondrial
precursor (delta-1 pyrroline-5-carboxylate dehydrogenase) lined,
through glutathione S-transferase, to IgG binding domains derived
from staphylococcal Protein A. The native pPGPrA becomes
irreversibly arrested, both in vitro and in vivo, while being
translocated across the outer and inner membranes of mitochondria.
During in vivo import of pPGPrA, the outer and inner membranes of
mitochondria become progressively "zippered" together, forming long
stretches of close contact. Affinity chromatography of solubilized
~zippered~ membranes has identified several core components of both
translocation channels. These proteins are specifically associated
with the arrested PGPrA intermediates. They include three GTP-
binding proteins, as well as all know essential elements of Tom and
Tim. The final goal is to determine how the newly identified GTP-
binding proteins participate (together with other Toms and Tims) in
distinct stages during mitochondrial protein import.

This project is of direct health-related significance, since
perturbation of mitochondrial function is associated with specific
mitochondrial disease, aging, and certain neurodegenerative
disorders. There is strong evidence that the constituents of
mitochondrial translocases and their role in protein import are
conserved between yeast and humans.
StatusFinished
Effective start/end date9/1/988/31/04

Funding

  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences: $185,546.00
  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences: $177,121.00
  • National Institute of General Medical Sciences: $180,140.00

ASJC

  • Filtration and Separation

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