protein import in mitochondria

What is the function of the mitochondrial presequence?

It targets nuclear-encoded proteins to mitochondria using an N-terminal amphipathic α-helix.

What is the structural feature of the mitochondrial targeting presequence?

One side hydrophobic and the other hydrophilic with positively charged residues.

Why does the presequence have a net positive charge?

It helps drive import across the inner membrane due to the membrane potential.

What experiment is commonly used to visualize mitochondrial targeting?

Fusion of the presequence to GFP.

Why is yeast commonly used to study mitochondrial import?

It is easy to genetically manipulate and yields pure mitochondrial preparations.

How does a cell-free mitochondrial import assay work?

Radiolabeled precursor proteins are mixed with isolated mitochondria to test translocation and cleavage.

What does low temperature do in mitochondrial import assays?

It traps intermediates with the signal stuck in the channel.

What does the DHFR folding experiment test?

Whether mitochondrial import requires the protein to be unfolded.

What effect does methotrexate have on DHFR?

It stabilizes DHFR in a folded conformation and prevents unfolding.

What happens when DHFR is stabilized during mitochondrial import?

The precursor becomes stuck and cannot fully translocate.

What conclusion comes from the DHFR import assay?

Mitochondrial import requires the protein to be unfolded.

What is the TOM complex?

The Translocase of the Outer Membrane responsible for initial protein entry.

Name key TOM complex components.

Tom20

What is the main pore-forming protein in the TOM complex?

Tom40.

What is the TIM23 complex?

The inner membrane translocase for matrix-targeted proteins.

What is the TIM22 complex?

An inner membrane translocase for carrier/multi-pass proteins.

What is the SAM complex?

The outer membrane complex that inserts β-barrel proteins.

What is the OXA complex?

The inner membrane insertase that inserts proteins from matrix to IM.

Describe the basic path of a matrix-targeted protein.

Presequence → TOM → TIM23 → MPP cleavage in matrix → folding.

What happens to the presequence once inside the matrix?

It is cleaved by mitochondrial processing peptidase (MPP).

What are the steps of TOM complex purification?

His-tag Tom22 → solubilize OM with gentle detergent → affinity chromatography → elute with imidazole.

Why do multiple proteins co-elute with His-Tom22?

They form a stable multi-protein TOM complex.

What does size-exclusion chromatography reveal about TOM?

All components elute together

What does reconstitution of TOM into liposomes show?

Purified TOM alone forms a functional translocation pore.

What happens when TOM is depleted from membrane fractions?

Import activity virtually disappears.

What does electrophysiology reveal about Tom40?

It behaves as a gated channel with open/closed states.

Why does Tom40 respond to voltage?

Signal peptides are positively charged and influence pore opening.

What is the approximate size of the Tom40 pore?

~20 Å

What did medium-resolution CryoEM show about TOM?

The core TOM complex forms a dimer with two Tom40 pores.

What did high-resolution CryoEM reveal?

Detailed arrangement of Tom40

What higher-order assemblies can TOM form?

Dimers and trimers