Mitochondrial Protein Transport

Post-Translational Transport to the Mitochondria

• The lecture describes the five steps of protein transport pathways.
• These steps are then identified in the mitochondrial pathway to understand transport.
• Experimental evidence is interpreted to support the model of transport.
• Apply the terms necessary in the transport of proteins to the mitochondria.

Characteristics of Mitochondria

• Bound by a double membrane with an interior and exterior membrane.
• Essential for ATP production.
• Proteins of the electron transport chain exist in the inner mitochondrial membrane.
• Possess their own genomes.
• Mitochondrial genes code for some of its proteins.
• Reproduce through fission.

Mitochondrial Structure

• Outer membrane surrounds the entire organelle.
• Inner membrane presents involutions called cristae that form distinct regions in the matrix.
• Cristae of the inner membrane increase the surface area for ATP synthesis.
• The region between membranes constitutes the intermembrane space.
• Mitochondrial regions are identifiable via electron microscopy.
• The inner membrane houses ATPase and the electron transport chain proteins
• The proteins convert nutrients into cellular energy (ATP)
• The structure increases the capacity to synthesize ATP.

Presence of Mitochondria in Cells

• Found in all cells, especially those needing a lot of ATP.
• Extensive mitochondria arrays reside in cardiac muscle cells that constantly provide ATP to fuel cardiac muscle contraction.
• Sperm cells require a lot of energy to maintain movement.
• Mitochondria form mitochondrial tubules that wrap around the axoneme of the sperm tail.
• Extensive mitochondrial networks are found in neural cells that require a lot of ATP.
• Mitochondria appear to form an elaborate network of tubules in neural cells.

Mitochondrial Dynamics and Genome

• Dynamic organelles change shape via fission and fusion.
• Mitochondria are in constant motion within the cytosol.
• They move, grow, and divide similarly to unicellular organisms.
• Contain multiple mitochondrial genomes.
• Undergo fission to produce two new mitochondria.
• Undergo fusion.
• Daughter mitochondria usually ends up with at least one mitochondrial genome during fission.
• Daughter mitochondria without a genome will degenerate and die.
• Mitochondria grow via the import of new proteins and genesis of new membrane.

Protein Destination and Origin

• Proteins are found in four locations within the mitochondria: outer membrane, inner membrane, intermembrane space, and matrix.
• Some proteins are coded in the mitochondrial genome and synthesized using mitochondrial ribosomes.
• Most mitochondrial proteins are coded by nuclear genes.
• Nuclear genes are transcribed in the nucleus and the mRNAs are translated in the cytosol by free ribosomes.
• Proteins are transported to the mitochondria in a post-translational pathway.

Testing Post-Translational Transport

• Hypothesis states that fully transported proteins can be transported into the mitochondria post-translationally.
• Mitochondrial protein synthesis is studied in a cell-free system.
• Polypeptides are combined with a mitochondrial signal sequence in a tube without ribosomes.
• Energized mitochondria get added first, followed by a protease which results with the proteins being protected.
• Energized mitochondria were not added and only the protease which resulted with the proteins being degraded.
• Fully translated proteins are transported into the mitochondria.
• Protein degradation is tested using an antibody to see if the protein localized within the mitochondria or through gel electrophoresis.

Mitochondrial Protein Transport Rules

• There must be a signal sequence.
• There must be a receptor for that signal sequence.
• There must be a translocation channel across the membrane.
• There must be a source of energy such as ATP hydrolysis.
• There must be a way of targeting proteins to different locations within an organelle.

Rules of Protein Transport to the Mitochondria: Signal Sequence

• The peptide signal sequence is known as the matrix-targeting motif.
• Located at the N-terminus, it contains 18 to 50 amino acids that form an alpha-helix.
• Specifically, it is an amphipathic helix (hydrophobic on one side, hydrophilic on the other).
• Positively charged hydrophilic residues and the hydrophobic residues are arranged in an alpha helix fashion.

Necessity of the Matrix Targeting Motif

• Hypothesis disruption of the amphipathic helix will inhibit the protein from reaching the mitochondria.
• Mutations disrupting the amphipathic nature of the helix will affect transport.
• Replacing hydrophobic residues with hydrophilic residues disrupts the helix.
• The matrix-targeting motif is required for protein transport to the mitochondria.
• Wildtype mitochondrial protein (with unmodified matrix targeting motif) are seen in the mitochondria.
• Mitochondrial protein with a mutant matrix-targeting motif remains in the cytosol.

Sufficiency of Matrix-Targeting Motif

• Matrix-targeting motif is needed for protein transport to the mitochondria.
• Adding the matrix-targeting motif to green fluorescent protein (GFP) re-localizes a protein from the cytosol into the mitochondria.
• Adding the motif results in the GFP no longer being diffuse, and can be seen in a punctate pattern.
• Localize a cytosolic protein into the mitochondria by simply adding the matrix-targeting motif.
• The matrix-targeting motif alone is sufficient for protein transport to the mitochondria.

Signal Receptor

• Matrix-targeting motif, is recognized by an import receptor that is embedded in the outer membrane of the mitochondria.
• The amphipathic helix of the matrix-targeting motif fits into the hydrophobic binding pocket of the import receptor.
• Hydrophobic residues of the amphipathic helix face into the hydrophobic pocket.
• The interaction facilitates recognition of the mitochondrial signal peptide sequence via the signal receptor.

Translocation Channel

• The translocation channel is also known as the general import pore (Tom40).
• The matrix-targeting signal then translocates from the import receptor into the general import pore.
• The protein is then shuttled through that translocation channel.
• The protein will continue straight into another translocon of the inner membrane (Tim44, Tim23, Tim17 complex), if targeted to the matrix.
• Two translocons are aligned at sites called contact sites.
• Proteins with a matrix-targeting motif move directly through both openings.

Requirement for ATP Hydrolysis

• Mitochondrial proteins are first translated in the cytosol then grabbed by cytosolic chaperone proteins (Hsc70).
• Hsc70 maintains the polypeptide unfolded to fit through the pores of the Tom and Tim translocons.
• Hsc70 function requires ATP hydrolysis.
• ATP hydrolysis is also needed in the mitochondria during protein transport by matrix Hsc70 proteins.
• The Hsc70 pulls proteins through the translocon by grabbing the unfolded proteins as they enter the matrix.
• This prevents the protein from moving backwards through the translocon.
• ATP hydrolysis drives a conformational change in Hsc70 and pulls the polypeptide into the matrix.
• Each Hsc70 protein binding creates space for another Hsc70 protein to bind.

Processing of Proteins Inside Mitochondria

• An unfolded protein must be processed after entering the mitochondria.
• Matrix processing protease removes the N-terminal matrix-targeting sequence.
• If the sequence is not removed, then prevents protein folding.
• Once the matrix-targeting sequence is removed, Hsc70 proteins assist in protein folding.

Protein Destination in Mitochondria

• Proteins destined for the mitochondrial matrix reach mitochondrial membranes.
• Transport to membranes requires a stop-transfer- sequence in addition to an N-terminal matrix-targeting sequence.
• Hydrophobic sequences are located in the interior of the protein sequence, and target the protein to the inner membrane.

Protein Transport to the Inner Mitochondrial Membrane

• The initial steps for transport of a protein into the inner membrane mirrors the transport into the matrix.
• N-terminal motif gets recognized by the import receptor and translocated through the Tom and Tim translocons.
• N-terminus of the protein appears in the mitochondrial matrix.
• The translocon of the inner membrane will then recognize the stop-transfer-sequence for the protein.
• The stop transfer sequence forms a hydrophobic alpha helix that stops translocation and directs transfer of the polypeptide out of the translocon into the inner membrane.
• The inner membrane translocon opens laterally and stops the protein from being pulled through the translocon.
• This exposes the stop-transfer-sequence is expose to the hydrophobic environment of the inner membrane.
• The hydrophobic alpha helix diffuses and the protein is embedded.
• Once the polypeptide has left, the Tim translocon closes

Stop-Transfer Sequence

• Stop-transfer-sequence is needed for transport into the inner membrane, but is not needed to get into the mitochondria.
• Adding only the stop transfer sequence will make the GFP protein stay in the cytosol.
• The stop transfer sequence alone is not sufficient for getting into the inner membrane.
• Proteins also need a matrix-targeting sequence in order to enter the inner membrane.

Importance of Protein Unfolding

• The cytosolic protein DHFR gets tagged with a matrix-targeting motif and a spacer sequence.
• Hsc70 proteins keep the DHFR protein unfolded, allowing it to be transported into the mitochondrial matrix.
• A matrix-targeting-motif can be sufficient for transport.
• Methotrexate can maintain the folded conformation of DHFR, despite Hsc70.
• Matrix targeting motif are successful in transporting the protein and some of the protein gets pulled through the translocons.
• The spacer sequence spans the two translocons and the matrix- targeting sequence is cleaved but the DHFR is folded so it cannot get through.
• Methotrexate added makes the DHFR aggregate in the cytosol at the contact site and cannot enter.
• Protein unfolding is necessary for transport of proteins to the mitochondria.

Mitochondrial Protein Import

• Mitochondrial protein transport follows the 5 rules for protein transport and constitutes a crucial process for cells.
• Defects might be caused due to mutations in targeting signals, the import machinery, or deficiencies in the chaperone (Hsc70).
• Often associated with neurodegeneration, mitochondrial protein import defects lead to diseases that affect any system within the body.
• Cells carry out post-translational protein transport to the peroxisomes and mitochondria.
• Proteins are targeted to the chloroplast post-translationally using an N-terminal targeting motif.
• Proteins are also targeted to the nucleus using a C- terminal nuclear localization sequence.

Protein Targeting Conclusion

• Protein targeting to the mitochondria follows the rules of post-translational targeting.
• Essential rules include a signal sequence on the transported proteins.
• There is a receptor for the signal sequence.
• Translocation channels must be available.
• ATP hydrolysis is required.
• A targeting mechanism for membranes or the interior of the organelle must be in place.
• Polypeptides must be unfolded when transported into the mitochondria.