Mitochondrial Protein Import

Core Concept

Mitochondria contain their own small genome, but it encodes only a tiny fraction of the organelle's proteins; well over 95% of mitochondrial proteins are encoded by nuclear genes, synthesized on cytosolic ribosomes, and imported post-translationally. Because the precursor is made in the cytosol before import, it must be kept unfolded by cytosolic chaperones. Import is directed by an N-terminal presequence (matrix-targeting signal) that is recognized by receptors of the TOM complex in the outer membrane and threaded through the TIM23 complex in the inner membrane. Import of matrix proteins requires both the inner-membrane electrochemical potential (Δψ) — negative inside — and ATP hydrolysis by mitochondrial Hsp70 in the matrix. This is a fundamentally different mechanism from ER co-translational translocation and from nuclear gated transport.

Key Components

• Presequence (matrix-targeting signal): an N-terminal, ~15–50 residue amphipathic α-helix rich in positively charged and hydroxylated residues; cleaved by the mitochondrial processing peptidase (MPP).
• Cytosolic chaperones (Hsp70/Hsc70): keep the precursor unfolded and competent for import.
• TOM complex: outer-membrane translocase; Tom20/Tom22 receptors bind the presequence; Tom40 forms the channel.
• TIM23 complex: inner-membrane translocase for matrix proteins (and some inner-membrane proteins); contains Tim17/Tim23/Tim50.
• TIM22 complex: inserts polytopic inner-membrane proteins (carrier proteins) in a Δψ-dependent manner.
• Mitochondrial Hsp70 (mtHsp70 / mortalin): the matrix chaperone that drives import by ATP-dependent binding of the incoming chain.
• OXA complex: inserts proteins into the inner membrane from the matrix side.

Mechanism / How It Works

1. A cytosolic ribosome completes the precursor; cytosolic Hsp70 chaperones keep it unfolded.
2. The positively charged presequence is recognized by Tom20/Tom22 and inserted into the Tom40 channel of the TOM complex.
3. The presequence is transferred to the TIM23 complex. Because the inner membrane's Δψ (negative on the matrix side) exerts an electrophoretic pull on the positively charged presequence, translocation across the inner membrane is driven forward.
4. In the matrix, mtHsp70 binds the emerging chain; repeated ATP-dependent binding (the "Brownian ratchet" or pulling model) biases the chain's thermal motion inward, completing translocation.
5. The MPP cleaves the presequence, and the mature protein folds with the aid of matrix chaperones (Hsp60/Hsp10 chaperonin).
6. Proteins destined for the inner membrane or intermembrane space use additional signals and the TIM22/OXA pathways (e.g., a second hydrophobic signal after the presequence for some inner-membrane proteins).

Energy and Directionality

Mitochondrial import uniquely requires two energy sources: (1) the electrochemical potential Δψ across the inner membrane, which drives the positively charged presequence through TIM23, and (2) ATP, hydrolyzed by both cytosolic Hsp70 (to keep the precursor unfolded) and matrix mtHsp70 (to drive vectorial translocation). Depleting Δψ with uncouplers (e.g., CCCP) blocks import at the TIM23 step, distinguishing mitochondrial import from ER import, which needs neither Δψ nor post-translational unfolding.

Experimental Evidence / Technique

The post-translational nature of import was shown by in vitro import assays: purified, denatured (or chaperone-bound) precursors imported into isolated mitochondria only after protein synthesis was complete, and import was blocked by uncouplers that collapse Δψ but not by inhibitors of cytosolic protein synthesis. Protease-protection experiments demonstrated staged import (outer vs. inner membrane) by using swelling to rupture the outer membrane. Yeast temperature-sensitive mutants (e.g., in TOM and TIM genes) identified the translocase components. These findings established the presequence/TOM/TIM/mtHsp70 model.

How it works

1. A cytosolic ribosome completes the precursor; cytosolic Hsp70 chaperones keep it unfolded.
2. The positively charged presequence is recognized by Tom20/Tom22 and inserted into the Tom40 channel of the TOM complex.
3. The presequence is transferred to the TIM23 complex. Because the inner membrane's Δψ (negative on the matrix side) exerts an electrophoretic pull on the positively charged presequence, translocation across the inner membrane is driven forward.
4. In the matrix, mtHsp70 binds the emerging chain; repeated ATP-dependent binding (the "Brownian ratchet" or pulling model) biases the chain's thermal motion inward, completing translocation.
5. The MPP cleaves the presequence, and the mature protein folds with the aid of matrix chaperones (Hsp60/Hsp10 chaperonin).
6. Proteins destined for the inner membrane or intermembrane space use additional signals and the TIM22/OXA pathways (e.g., a second hydrophobic signal after the presequence for some inner-membrane proteins).

Common confusions

• "Mitochondrial proteins are made inside mitochondria." — Almost all are nuclear-encoded and cytosolically synthesized; only ~13 (in humans) proteins are made on mitochondrial ribosomes.
• "Mitochondrial import is co-translational like the ER." — It is post-translational; the precursor is fully made (and kept unfolded) before import.
• "ATP alone powers import." — Import needs Δψ too; collapsing Δψ with an uncoupler blocks import even with abundant ATP.
• "Mitochondrial import uses Ran." — No; Ran is for nucleocytoplasmic transport. Mitochondria use presequence + TOM/TIM + Hsp70 + Δψ.
• "The presequence stays on the protein." — It is cleaved by MPP after import (unlike an NLS, which is retained).

Quick review

• Nuclear-encoded, post-translational import via presequence.
• TOM (outer) → TIM23 (inner) → matrix; TIM22/OXA for inner-membrane proteins.
• Energy: Δψ (electrophoretic pull) + ATP (cytosolic and matrix Hsp70).
• MPP cleaves the presequence; Hsp60/Hsp10 assist folding.
• Uncouplers block import — a diagnostic feature.
• Distinct from ER translocation and nuclear gated transport.