M4L2 - Post-Translational Targeting to Mitochondria

Characteristics of Mitochondria

• 1. Bound by a double membrane

• 2. Primary site of ATP production

  • Proteins of ETC are found in inner mitochondrial membrane

• 3. Has their own genomes 

  • Mitochondrial genes code for a few of the proteins found in the mitochondira 

• 4. They can reproduce by binary fission 

Internal Compartmentalization of Mitochondria

• Has outer membrane

• Has inner membrane with involutions to create cristae

  • Inc SA where ATP synthesis occurs from nutrients

  • This structure increases the capacity of mitochondria to create lots of ATP

• Between membranes: Intermembrane space 

• Right: TEM image 

Mitochondria Distribution in Different Cell Types

• Cells requiring a lot of ATP will have more mitochondria

Cardiac Muscle: (Top Left)

• Actin bundles (green) required for continuous muscle cell contraction

• Arrays of mitochondria present (red) throughout the cells

• They constantly provide ATP to fuel cardian muscle contraction

Sperm Cell: (Top Right)

• Moves rapidly, so needs a lot of E to maintain this movement

• TEM image: Flagellar Axoneme Middle Cross-Section

  • Surrounding it is mitochondria

  • Forms mitochondrial tubules that wrap around the axoneme

Neural Cell: (Bottom Left) 

• Needs a lot of E to function 

• Nucleus (Blue labelled with DAPI) 

• Actin (Green labelled with phalloidin) 

• Mitochondria (Red labelled with antibody to mitochondrial-specific protein) 

• Mitochondria doesn’t show up at punctate dots but as a network of tubules 

Mitochondria Cell Dynamics

• Change shape

• Undergo fission/fusion 

• Always moving around 

• Grows

Mitochondria Biogenesis

• Requires protein synthesis

• Contains much of its own genome

• A single mitochondria can divide into 2 by fission 

  • Each daughter usually ends up with one genome copy at least 

  • if not, it would die 

• 2 mitochondria can undergo fusion 

Destinations of Proteins Within Mitochondria (4)

1. Outer membrane

2. Inner Membrane

3. intermembrane space 

4. matrix of mitochondria 

Where do Mitochondrial Proteins Come from?

• Some are encoded in the mitochondrial genome

  • Synthesized using mitochondrial ribosomes 

• Majority are coded by nuclear genes 

  • Genes transcribed in nucleas

  • mRNA translated in cytosol by free ribosomes 

  • Proteins transported to mitochondria by pathway 

Post-Translational Protein Transport to Mitochondria Evidence

• Hypothesis: If fully translated proteins can be transported, then proteins in the presence of mitochondria will move into it 

Experiment:

• Follow the protein synthesis in cell-free system 

• Tube 1: Polypeptides with mitochondrial signal sequence (red) 

• Experiment 1:

  • Add energized mitochondria first 

  • Then add protease 

• Experiment 2: (Bottom)

  • Just add protease but no mitochondria

Result:

• In experiment 1, the proteins are safe from degredation as they’re transported to mitochondria

• In experiment 2, no mitochondria so proteins degrade

• Conclusion: Fully translated proteins can be transported into mitochondria

Rule 1: Protein Transport to Mitochondria

• Matrix-Targeting Motif:

  • Peptide signal sequence

  • Found on N-terminus

  • 18-50 amino acid long peptide

  • Forms an a-helix that is amphipathic

• Red: (+) residues that are hydrophilic

• Yellow: Hydrophobic residues

• Regular arrangement of hydrophobic/philic residues allowing them to be on opposite surfaces when folded 

Is Matrix-Targeting Motif Necessary for Protein Transport to mitochondria?

• hypothesis: if the amphipathic helix is disrupted, a mitochondrial protein will not go to the mitochondria

• test by

  • creating mutations disrupting the amphipathic nature 

  • eliminating the motif 

Experiment:

• Hydrophobic residues replaced by hydrophilic ones

• Top Image: wild-type mitochondrial protein (unmodified matrix-targeting motf) 

  • Present in mitochondria

  • Detected using antibody to the protein (green)

• Bottom Image: Mutant matrix-targeting motif

  • mitochondrial protein remains in cytosol

• Conclusion: Yes, matrix-targeting motif is necessary

Is Matrix-Targeting Motif Sufficient for Protein Transport to mitochondria?

• Hypothesis: If matrix-targeting motif is added to GFP, then it will go to mitochondria

• Result: The GFP with matrix-targeting motif went to the mitochondria as it was seen in a punctate pattern 

• Conclusion: Yes it is sufficient 

Rule 2: Protein Transport to mitochondria

• Import Receptor

  • A signal receptor recognizing matrix-targeting motif 

  • Embedded in out membrane of mitochondria

• How does it recognize the matrix-targeting motif? 

  • Amphipathic helix of the motif fits into the hydrophobic binding pocket of the receptor 

  • All hydrophobic residues of the amphipathic helix are facing the hydrophobic pocket 

Rule 3: Protein Transport to mitochondria

• General Import Pore (AKA Tom40) 

  • Translocation channel 

• When bound to import receptor, the matrix-targeting sequence is translocated to the general import pore 

• The protein is shuttled through that translocation channel 

• If targeted to the matrix, it will continue into another translocon of the inner membrane 

  • The Tim44, Tim23, Tim17 complex 

• At certain contact sites, the two translocons are aligned (Tim/Tom)

  • Allows for direct movement of protein going to matrix

Rule 4: Protein Transport to mitochondria (2)

Instance 1:

• Mitochondrial proteins first are translated in cytosol

• They must remain unfolded to fit through Tom and Tim translocons

• So they’re grabbed by cytosolic chaperone proteins (Hsc70) to keep them unfolded

• Hsc functions requires ATP hydrolysis

Instance 2:

• ATP hydrolysis needed in mitochondria during protein transport

• Matrix Hsc70 grab unfolded protein as it enters matrix

• Prevents the protein from moving backwards

• ATP hydrolysis conformationally changes Hsc70 that will pull the protein into the matrix 

• Another Hsc70 comes to bind, enabling the full protein to be pulled through 

Protein Folding in Mitochondria (after transport)

• Matrix processing protease removes N-terminal matrix-targeting sequence 

  • Otherwise the protein won’t fold 

• Hsc70 proteins then further assist it in folding 

Rule 5: Protein Transport to mitochondria

• To transport to inner membrane there needs to be

  • N-terminal matrix targeting sequence

  • stop-transfer sequence 

• Stop-transfer Sequence

  • Hydrophobic sequence

  • found in the middle of the protein sequence 

  • Helps the target protein go to the inner membrane as it gets stuck in lipid membrane bc hydrophobic 

Protein Transport to Inner Mitochondrial Membrane

• Initially same as transport to the matrix

  • N-terminal motif recognized by import receptor 

  • N-terminus translocated through the Tom and Tim translocons 

• The N-terminus of mitochondrial protein does go to the mitochondrial matrix 

  • However, the Tim also recognizes the stop-transfer sequence 

• Stop-transfer sequence forms an hydrophobic alpha helix that does 2 things

  • 1. Stop the translocon so protein is no longer pulled through 

  • 2. Directs the transfer of the protein out of the translocon and into the inner membrane 

• The translocon open laterally (sideways) 

  • Creates an opening exposing the stop-transfer sequence to the hydrophobic env of inner membrane 

  • The proteins then imbeds into the inner membrane 

Is the stop-transfer sequence necessary for inner membrane transport? 

• Yes

• The protein would still get into the matrix due to the matrix-targeting sequence

• The protein won’t be able to embed itself into the inner membrane though

• So it’s necessary for transport to the inner membrane but not for transport to matrix

Is the stop-transfer sequence sufficient for inner membrane transport? 

• Tagging a cytosolic protein (Ex. GFP) with the stop-transfer sequence 

• If you add JUST the stop-transfer sequence, the protein stays in cytosol 

• So it’s not sufficient for transport to inner membrane

  • It’s necessary though 

Is unfolding necessary for protein transport to mitochondria? (DHFR)

• Target cytosolic protein for transport (DHFR shown in blue) 

• Tag it with matrix-targeting motif (red) and add a spacer sequence (black)

  • Length of 2 translocons 

• Presence of Hsc70 keeps DHFR unfolded allowing it to be transported into the mitochondrial matrix 

• So any unfolded protein is getting through, so long as it has the matrix-targeting motif atleast which is sufficient for transport

Is unfolding necessary for protein transport to mitochondria? (DHFR and Methotrexate)

• Methotrexate maintains folded confromation of DHFR despite Hsc7- 

• Matrix-targetinig motif is still sucessful in getting the protein to mitochondria 

  • Some of the protein is pulled through translocoon 

  • The spacer sequence gets through 

  • the matrix-targeting sequence gets cleaved

• Since DHFR is folded, it cannot get through 

  • Protein unfolding is therefore necessary for transport of proteins into mitochondria

Defects in Mitochondrial Transport

• Causes: 

  • Mutations in target signals 

  • Mutations disrupting import machinery 

  • Deficiencies in the chaperone Hsc70 

• Can effect any system in body 

  • Often associated with neurodegeneration though 

Post-Translational Targeting to other Organelles

• Chloroplast: 

  • Uses N-terminal targeting motif 

• Nucleus

  • Uses C-terminal nuclear localization sequence