Lecture 1 + 2 "Principles of (protein) transport"

On cytosolic ribosomes

Proteins need to be delivered to the place of function

• domains with the cytsol

• into diffrent rganelles

• into different membanes

• into the extracellular space

> 50 % of all proteins have to pass the membrane

• mRNA based

• protein signal based

Part of the polypetide chain anf in some cases is cleaved after succesful transport and translocation

Depends on signal composition and localization on targeted organelle

• Delivery of one protein to more than one cellular destination

• isolation of organelles

• WB or MS/MS

• GFP tagging

• semi-quantification possible

• analysis of kinetics possible

• impact of Targetting omitted

• many reporter possible

• impct of taretting detectable

• analysis of kinetics possible

• ER, Nucleus

• Thylakoids,Chloroplasts

• Mitochondira, Peroxisomes

• Bacteria

• non-cleavable signal anchor sequences (SAS)

• C-terminal tail anchor sequences (TAS)

• cleavable signal peptides (SP)

• RNA protein complex that recognized emerging polypeptides

-Yes, across archea, bacteria ad eukaryotes

1. Signal recognition by the SP54

2. Elongation arrest by Alu domain

3. Targetting complex formation

4. Docking of RNC to translocon

5. Signal release into translocon by the GTP hydrolysis

• Bind at the ribosomal exit tunel

• ATP hydrolysis by SecA transfers the signal unto the SecYEG channel

• ransferred across the periplasm with the help from chaperones

• BamA/OMP85

• binds to membrane (PG/MGDG)

• Recognition of substrate (ß-strand) release from mebrane

• after reales from membrane intraction with Omp85

• After interaction with Omp85 release of substrate

• Recognition of free substrate by Omp85

• though Phe/Gly (FG) domain-controlled barrier

• nucleocytoplasmic transport

• size selevtion of about 5 nm for molecules not interacting with FG

• phase seperation & FG hydrogel formation through “cohesive” interactions - weak and driven by hydrophobicity of FG motifs

• FG network requiered for the selective transport by NTF (nuclear transport factor)

• Nuclear localization signal (NLS)

  • specific for IMPORTIN

  • classical NLS (K-K/R-X-K/R)

  • basic PY-NLS

    • K-X2-K/R-K/R-X2-3-R/K/H-X2-5-PY

  • hydrophobic PY-NLS

    • phi-G/A/S-phi-phi-phi-X2-3-phi-x-phi-PY

• Nuclear export signal (NES)

  • specfic for EXPORTIN interaction

    • helical phi-X2-3-phi-X-2-3-phi-X-phi

• specific for the receptor

• partially defined by consensus motif

Karyopherin-mediated transport involving factors such as NTF2

• Import starts when a cargo protein with an NLS binds an importin in the cytoplasm.

• The importin-cargo complex moves through the nuclear pore by interacting with FG-repeat nucleoporins.

• In the nucleus, Ran-GTP binds importin and makes the cargo let go.

• The cargo stays in the nucleus; the importin is recycled back to the cytoplasm.

• In the cytoplasm, Ran-GAP turns Ran-GTP into Ran-GDP, which resets the system.

• Export works the opposite way: an exportin binds cargo only in the nucleus.

• Ran-GTP helps exportin bind cargo and form the export complex.

• The complex moves to the cytoplasm, where Ran-GAP triggers GTP hydrolysis.

• Hydrolysis breaks the complex apart and releases the cargo in the cytoplasm.

• Ran-GDP is transported back to the nucleus and recharged to Ran-GTP.

• The Ran-GTP gradient is what gives the transport direction: high Ran-GTP in the nucleus, high Ran-GDP in the cytoplasm.

• Pex5 binds the cargo in the cytosol.

• The receptor-cargo complex docks at the peroxisome membrane.

• The cargo is imported into the matrix.

• Pex5 is recycled back to the cytosol.

• Slide 40 also shows de novo formation from the ER.

Cargo docks at the peroxisome membrane and enters the matrix.

• The receptor is recycled after cargo release.

• Existing peroxisomes can grow and divide.

• New peroxisomes can also form de novo, with ER and sometimes mitochondria involved.

• Cargo selection: In the cytosol, the receptor PEX5 recognizes a peroxisomal targeting signal, usually the PTS1 at the cargo’s C-terminus.

• Docking: The PEX5–cargo complex binds the peroxisomal docking/translocation machinery on the membrane.

• Membrane passage: PEX5 and cargo move into the peroxisome through a translocation step; peroxisomes are unusual because they can import folded proteins.

• Cargo release: Once inside, the cargo is released into the peroxisomal matrix.

• Recycling signal: PEX5 then becomes modified, typically by monoubiquitination, which marks it for return.

• Export/recycling: The ubiquitin ligase complex helps extract PEX5 back to the cytosol through a retrotranslocation step.

• Reset: After removal from the membrane, PEX5 is deubiquitinated and reused for another import round.

• Why it is a cycle: The same receptor keeps shuttling between cytosol and peroxisome, so the process repeats continuousl

• mitochondria: TOM complex

• chloroplasts: TOC complex

• TOM 40: Porin/VDAC family

  • uneven number of beta strands

-Toc75: Omp85 family

• even number podra domain

1. TIM23 closed

2. Priming

3. TIM23 open

4. Binding to respiratory chain

5. a. Sorting: lateral release and IM integration

   or

   b. Primig for Matroix

6. Motor assembly

7. Active motor

8. Matrix translocation

Loop = (TIM) 22

1. Precursor bound to chaperones (Hsp70,90)

2. Binding of precursor-chaperone complex to Tom70 oligomer

3. a Translocation across OM and binding to Tim9-Tim10

   b. Tethering to Tim9-10-12

4. Insertion into TIM22 complex

5. Assembled dimer

1. Translocation across OM

2. Recognition by MIA40

3. Precursor oxidation by MIA40/Erv

4. Oligomeric assembly

5. Reoxidation by Erv1

6. Transport of electrons to respiratory chain

1. N-signal anchored

2. c-tail anchored

3. internal signal

4. multiple transmembrane segments

• Mitochondrial protein import is linked to mitochondrial shape and organization, not just transport.

• The import machinery helps bring in proteins needed for mitochondrial function and maintenance.

• So the big idea is a two-way connection: import affects morphology, and morphology affects import

• Chloroplast proteins are made in the cytosol and imported after translation.

• They enter through TOC and then pass through TIC.

• The import pathway is energy-dependent and involves additional processing/folding control

1. Co-translational

2. post-translational

3. retro-translational