MCB 2210 L16: Mitochondria, Chloroplast and Peroxisome Targeting

Slides 1-30

Explain the evolutionary ancestry of mitochondria and chloroplasts

• Evolved from ancient bacterial eaten by eukaryotic cell

  • Inner + Outer membranes

    • Inner membrane = original bacterial membrane

    • Outer membrane = eukaryotic cell membrane (ate)

Describe fission and fusion and how they relate to mitochondria and chloroplasts

• Mitochondria ≠ static → constantly Δshape through 2 processes

  • Fission = divide into 2 → cell division

    • Ensures 2 daughter cells have mitochondria

    • Remove damaged sections of mitochondrial network

  • Fusion = 2 fuse tg → long organelle

    • Share resources (proteins, healthy DNA) → repair/support underperforming sections of network

Note: chloroplasts undergo fission but NOT fusion

Describe structure, function & location of mitochondria

• Structure

  • 2 membranes

    • Outer membrane

    • Inner membrane = folded in → INCREASE surface area

  • Matrix = innermost space

  • Intermembrane space = space b/w 2 membranes

  • Shape = cylindrical + dynamic

    • Can fuse to tubes + Δ shape

    • Role of Δ morphology (shape) NOT understood

• Function = produce ATP for cellular energy

  • Cytoplasm → Glycolysis = breaks down glucose → 2 ATP + pyruvate

  • Mitochondria = Oxidative metabolism (Citric acid/Krebs cycle) = pyruvate → mitochondria → broken into 30+ ATP

• Location = throughout cytoplasm

  • Sometimes in areas where more energy is needed (muscle cells)

  • Associated w/ microtubules → move along microtubules through molecular motors

Describe structure + function + location of chloroplasts

• Structure

  • 2 membranes

  • 3rd membrane internal layer in thylakoids

• Thylakoid = tiny, sac-like membranes stacked inside plant chloroplasts

  • Inside = Thylakoid space/lumen

  • Outside = matrix = stroma (fluid in chloroplast surrounding thylakoids)

• Location of machinery:

  • Thylakoid membrane → photosynthetic machinery

  • Stroma → carbohydrate synthesis machinery

• Function

  • Use light energy to produce ATP

  • ATP → carbohydrate synthesis (glucose)

How are mitochondria involved in ATP production?

Convert pyruvate (product of glycolysis) → ATP

Describe the three steps required for ATP synthesis in mitochondria

1. Pyruvate → mitochondria → Citric acid cycle → NADH

2. NADH drops of e^- @ Electron Transport Chain (ETC)

   1. Pumps protons out → pressure gradient

3. Protons rush through ATP synthase → spin → ATP synthesis

What do chloroplasts use to generate proton gradient for ATP synthesis?

Use light energy

Where are proton gradients set up in mitochondria and chloroplasts?

Mitochondria → inner membrane

Chloroplast → thylakoid membrane

Both drive ATP synthesis in matrix/stromaZ

What are the 2 types of proteins found in inner and outer membranes of mitochondria? Describe how small molecule transport is achieved through these proteins.

• Beta-barrel channels = type of pore-forming protein structure found in outer membranes of bacteria, mitochondria, & chloroplasts = barrel made of twisted beta-sheets

  • Porins = specialized, barrel-shaped transmembrane proteins → form water-filled channels in outer membranes of bacteria, mitochondria, & chloroplasts

    • Allows small molecules + ions to move b/w intermembrane space & cytoplasm (through outer membrane)

• Transporters = specialized proteins (selective channels/carriers) located in cell membranes → selective

  • Located in inner membrane

  • Selectively moves small molecules across inner membrane in/out of matrix

    • Examples:

      • ADP/ATP exchanger = protein in inner mitochondrial membrane → ADP into mitochondria, ATP out mitochondria

      • Pyruvate/H⁺ cotransporter = protein complex in inner mitochondrial membrane

        • Pyruvate import to matrix

        • Final product of glycolysis → mitochondria for Citric Acid Cycle

      • Phosphate/H⁺ cotransporter (PiC) = protein in inner mitochondrial membrane

        • Phosphate (P_i) import

          • P_i → ATP synthase → ATP synthesis

Describe the mechanism of ATP synthase + experiment that was used to prove it spins

Rotary mechanism = γ subunit (central shaft) of ATP synthase rotates → converts H⁺ gradient energy → chemical energy in ATP

Scientists attached fluorescent actin filament to γ subunit

• Enzyme + energy → actin filament = spinning under microscope

• Enzyme = anchored to glass using His-tag + Ni-NTA coating

  • Kept in place as enzyme spun

What is special about mitochondria and chloroplasts specifically?

Have their own genomes (circular)

• >90% of mitochondria proteins encoded by nuclear DNA

• Mitochondria = mixture of proteins encoded by nuclear + mitochondrial genes

  • Some enzymes = mixtures of nuclear + mitochondrial subunits

  • Δ tissue → Δ mitochondria

  • Mitochondrial genomes of different species = overlapping genes

    • Absent in mtDNA → nuclear DNA

• Certain amount of genes → functioning mitochondria

  • Nuclear/mitochondrial genes depend on species

• Chloroplast = mixture of proteins encoded by nuclear + chloroplast genes

ATP Synthase is described as a ____________

Chimera = describes something made of different origins

• ATP synthase = protein subunits from 2 different places:

  • Mitochondrial DNA

  • Nuclear DNA

Describe mitochondrial protein synthesis pathways from mtDNA and nuclear DNA.

• mtDNA:

  • Protein = encoded by mtDNA → synthesized in matrix w/ mitochondrial ribosomes

    • 13 mtDNA-encoded proteins

• Nuclear DNA:

  • >90% mitochondrial proteins = encoded by nuclear DNA → synthesized in cytoplasm

    • ~1000 nuclear-encoded proteins

    • Nucleus → mRNA → protein by cytoplasmic ribosome

      • Pass through 2 membrane proteins to matrix

        • TOM = Translocase of the Outer Membrane

        • TIM = Translocase of the Inner Membrane

Describe protein localization to different mitochondrial compartments (matrix, inner membrane, outer membrane, intermembrane space). What signal is used?

Nuclear encoded proteins = targeting signal

• N-terminal amphipathic α-helix + (+) charged amino acids= helix w/ hydrophobic + hydrophilic sides

  • Necessary & sufficient → import → mitochondria

  • Signal = bound by receptor protein of outer mitochondrial membrane

• Post-translational transmembrane transport = process where protein synthesis → cytosol BEFORE transport across mitochondrial membrane

Mitochondrial-encoded proteins ≠ targeting signal

• Signal NOT NEEDED

• Synthesized in matrix

Describe the experimental paradigm used to prove protein transport mechanism to mitochondria.

1. Cytoplasmic ribosomes → synthesize yeast mitochondrial proteins (test tube) + N-terminal amphipathic α helix (targeting sequence)

2. Yeast mitochondria → test tube

   1. Proteins = taken up into mitochondria

   2. Targeting sequence = removed + degraded

3. Trypsin (protease) → test tube

   1. Proteins = intact bc sequestered/hidden inside mitochondria

4. Trypsin → test tube W/O mitochondria

   1. Proteins = degraded (no protection)

1. What happens if you add ribosomes, mRNA, mitochondria, and trypsin simulatenously?

2. How is this different than ER microsomes?

1. Trypsin → digest protein as soon as protein = synthesized

   1. No proteins imported safely

2. Difference in when import occurs

   1. Mitochondrial import = post-translational

      1. Mitochondria → entire protein digested

   2. ER import = co-translational

      1. ER → part of protein safe inside ER

Describe transport of nuclear-encoded proteins across the inner & outer membranes of mitochondria

TOM (Transporter Outer-Membrane mitochondria) = outer membrane pore complex + receptor for signal

• Protein transport → intermembrane space

• Protein = all possible protein destinations in mitochondria (outer membrane, inner membrane, intermembrane space, matrix)

TIM (Transporter Inner-Membrane mitochondria) = inner membrane pore complex

• Protein transport → matrix

OXA Complex = inner membrane protein that inserts protein into matrix

• Protein transport → matrix

Protein exposed to matrix → targeting signal cleaved off

Describe transport of proteins into matrix

Occurs across both membranes @ once

• Cytosolic chaperones keep protein unfolded

• Import receptor + TOM40 Complex = recognize signal sequence

• Protein transported through TOM40 complex → TIM44-TIM23/17 complex @ contact site

  • Contact site = specialized region where outer + inner membranes are tightly tethered to facilitate transfer of precursor proteins

• Matrix chaperone = binds protein + pull unfolded proteins across inner membrane → matrix

• Matrix protease = cleaves signal sequence

What are the 3 different pathways to the Mitochondrial Inner Membrane?

• Path A = Stop-Transfer Route (most common for single transmembrane protein)

  • N-terminal matrix-targeting sequence + stop-transfer anchor sequence

  • Protein → TIM23 complex → stop-transfer sequence = “stuck” in inner membrane

  • Complex = opens laterally → protein embedded in membrane

• Path B = Oxa1-Mediated Route (used by proteins → “re-inserted” into membrane from matrix side)

  • TOM/TIM23 pathway: Protein imported → matrix

  • Oxa1 = protein in inner membrane = inserts proteins into mitochondrial membrane

    • Recognizes protein & inserts back into inner membrane

  • Pathway for (nuclear + mitochondrial)-encoded proteins

• Path C = Multi-transmembrane Route (Multipass proteins) → Ex. ATP/ADP antiporter

  • NO N-terminal targeting sequence + HAS internal targeting sequences

    • Internal targeting sequences = acts as signal sequence + anchor in membranes

  • Bound by small chaperones in intermembrane (Tim9+10) → prevent folding/clumping

  • Enter matrix through TIM22 Complex (NOT TIM 23)

What are 2 pathways that proteins are targeted to intermembrane space?

• Path A = Protease cleavage

  • Protein enters inner membrane through Tim23/17 complex

  • Protease cleaves protein → released into intermembrane

• Path B = Direct entry

  • Protein passes through outer membrane

  • Protein stays in intermembrane space

  • Does NOT try to enter inner membrane

Explain protein targeting to chloroplast

• Similar to mitochondria

  • Chaperones = assist post-translation import (unfolded)

  • Stroma targeting signal = N-terminal amphipathic helix

    • Protein → outer membrane → bind to receptor

  • Transport occurs through TOC + TIC (similar to TOM + TIM)

  • Directed to different areas through secondary signals + complexes

  • Signal sequence = cleaved once protein → stroma

• Thylakoid → 4 routes, use thylakoid targeting sequence

• Plant cells = mitochondria + chloroplasts

  • Membrane receptors MUST be able to tell signals apart

    • Same signal → transported to both

    • Different signals → each organelle

    • Poorly understood

Peroxisomes + 2 mechanisms to multiple

Peroxisomes = organelles that specialize in oxidative reactions = degrade long fatty acid chains through β-oxidation = chimeras of ER + Mitochondria → bounded by single membrane bilayer

• β-oxidation = metabolic process of breaking down fatty acids inside mitochondria to generate ATP

  • Major byproduct = H₂O₂

1. De Novo Biogenesis = creating new peroxisomes

   1. ER + Mitochondria → “Pre-peroxisomal vesicles”

   2. Pre-peroxisomal vesicles + PEX16 + PEX3 fuse → “maturing peroxisome”

   3. Imports Peroxisomal Membrane Proteins (PMPs) + Matrix proteins (enzymes for β-oxidation) from cytosol

2. Growth & division

   1. Peroxisome imports more proteins + lipids → elongation

   2. Fission → 2 smaller peroxisomes

Import = post-translational

N-term/C-term peroxisomal targeting sequences (PTSs) → Unique cytoplasmic receptors

• Signals NOT cleaved after protein = inside

What are the 2 main pathways that bring proteins into peroxisome matrix? Define Zellweger Syndrome

1. PTS1 Pathway = uses Pex5 receptor (cytosolic)

   1. Pex5 binds → protein in cytosol → translocation complex on membrane → releases protein into matrix

2. PTS2 Pathway = uses Pex7 receptor (cytosolic)

   1. Transport proteins w/ different type of signal sequence

Zellweger Syndrome = mutation in Pex5 → empty peroxisomes

• Lethal, inherited disease

• Leads to cells w/ accumulated long fatty acid chains