Lecture 9: Protein Sorting (Gated Transport, Transmembrane transport)

Gated Transport, Transmembrane Transport To mitochondria, chloroplasts, peroxisomes

Animal Specific Cell-Component - Lysosome Matrix

Degradation of cellular components that are no longer necessary

Plant-Specific Cell Component - Vacuole

For storage and degradation (like animal’s lysosome)

Animal Specific Cell-Component: Peroxisome

Contain enzymes for oxidative reactions (some of these reactions can be done by mitochondria as well)

Post-Translational Protein Sorting

Protein are fully synthesized in the cytosol before sorting

Post-Translational Protein Sorting: Which ones remain unfolded and which ones remain folded?

Unfolded: mitochondria, chloroplasts

Folded: Nucleus, Peroxisomes

Co-Translational Protein Sorting

Proteins with ER Signal Sequence → associated with ER during protein synthesis

Gated Transport

Proteins moving to and from nucleus and cytosol

Transmembrane Transport

Requires protein translocators (proteins usually unfolded, except for peroxisomes)

Transport of protein across membrane

What is a Nuclear Pore Complex?

Transport of cargo go through NPC, transport in both directions (export and import)

Selective transport and free diffusions for small molecules

What is the Nuclear Pore Complex made up of?

made up of nucleoporin (many proteins)

What does cargo proteins have in order to help them get imported?

Nuclear Localization Signal → which binds with the Nuclear Import Receptor

For Import: NLS is rich in Lys and Arg

What does the Nuclear Import Receptor do?

Binds to the NLS of the cargo protein → binds to nucleoporins of NPC → transport into the nucleus

What is the use of the adaptor proteins?

Sometimes, some cargo proteins have a sequence that binds to the adaptor protein, and the adaptor proteins have the NLS, which binds to the Nuclear Import Receptor

What do the cargo proteins that want to be exported have?

Nuclear Export Signal → Nuclear Export Receptor binds to this part of the cargo

What does the Nuclear Export Receptor do?

binds to the NES of the cargo protein → binds to nucleoporins of NPC → transport into cytosol

Ran GTP and Ran GDP, where are they found?

Ran GTP (bound by GTP) - found in nucleus

Ran GDP (bound by GDP) - found in the cytosol

Ran GEF and Ran GAP what do they do?

Ran GAP: GTPase Activating Protein

• in the cytosol

• Cuts Ran GTP to Ran GDP

Ran GEF: Guanine Nucleotide Exchange Factor

• in the nucleus

• Exchanges Ran GDP to Ran GTP

NTF2 function?

Nuclear Transport Factor 2

• transports Ran-GDP into the nucleus

Ran-GTP and Ran-GDP Circulating

• Ran GTP in the nucleus → goes to cytosol → Ran GAP cuts the GTP into GDP → becomes Ran GDP

• Ran GDP is brought back into nucleus via NTF2 → Ran GEF exchanges GDP to GTP → becomes Ran GTP

Nuclear Import Mechanism

1. Cargo Protein’s NLS binds with Nuclear Import Receptor

2. Nuclear Import Receptor binds to nucleoporins in the NPC, and transports cargo into the nucleus

3. Bound by Ran-GTP → causes a release in cargo

4. Empty import receptor and Ran-GTP goes to cytosol

5. Ran-GTP is cleaved into Ran-GDP by Ran-GAP and Ran Binding Protein

6. Ran-GDP gets transported back into nucleus with NTF2

Nuclear Export Mechanism

1. Nuclear Export Receptor binds to cargo NES and Ran-GTP

2. Nuclear Export Receptor binds to nucleoporins of the NPC, and goes to cytosol

3. Ran-GTP is cut by Ran-GAP and Ran Binding Proteins (becomes Ran-GDP), where the cargo is released into cytosol

4. Empty nuclear export receptor gets transported back to nucleus

Example of Nuclear Import/Export - What is NFAT?

Nuclear Factor of Activated T Cells

Example of Nuclear Import/Export - NFAT: Import

high Calcium ions = encountered bacteria/virus: Nuclear Import to enable transcription

• Phosphatase cuts 3 Phosphates and blocks the export signal → this enables the import signal

• Imported into the cell = gene transcription activated

Example of Nuclear Import/Export - NFAT: Export

low Calcium ions = nothing to attack: Nuclear Export

• Protein kinase adds three Phosphates → changes shape

• This exposes nuclear export signal

• Leaves nucleus

Transmembrane Transport: Sorting to Mitochondria and Chloroplasts - Protein is translated ____ and is folded or unfolded?

Protein is fully translated in the cytosol, and when importing into organelle, it is unfolded

Transmembrane Transport: Sorting to Mitochondria and Chloroplasts - How do proteins remain unfolded?

hsp70 chaperones bind onto the unfolded proteins in the cytosol, and prevent it from folding

What do proteins that need to be transported to mitochondria and chloroplasts have?

N-terminus amphipathic alpha-helix → to bind to the transmembrane receptor

Transporting into Mitochondria mechanism

TOM (Translocase of outer membrane) and TIM23 (Translocase of the Inner membrane) used

• protein’s N terminal amphipathic alpha helix sequence is recognized by TOM

• Binds to TOM → moves to TIM23

• Protein moves into matrix

• N-terminal amphipathic alpha helix sequences are cleaved by signal peptidase

• Protein further folded

Transporting into Chloroplast mechanism

TOC (Translocase of outer chloroplast membrane) and TIC (Translocase of the inner chloroplast membrane) used

• Precursor protein has a N-terminal amphipathic alpha helix that is recognized by TOC

• TOC passes protein onto TIC, protein is now inside the stroma

• The N-terminus amphipathic alpha helix is cleaved off

• Once cleaved off, exposes thylakoid signal sequence

• Transported into the thylakoid → cleave off sequence

Are proteins folded for peroxisomal proteins? What target signals do they have?

Proteins for transporting into peroxisome is folded

Peroxisomal targeting signal = 3 amino acids (SKL) in the C terminus

Transporting into Peroxisome mechanism

• Protein’s C terminus SKL sequence binds to soluble receptor → goes to docking protein

• Docking protein tranferrs protein to translocator

• Translocator brings the protein in

• Does not cut the SKL sequence