Baumeister Protein Sorting

• a synthetic protein needs to contain all the signals and information for targeting it to the correct site of function e.g. receptor to plasma membrane or DNA polymerase to nucleus

• depending on the transport mechanism you need to keep in mind that it might need to be unfolded

• many sorting problems are associated with diseases

• the putative function of newly discovered proteins can be interpreted with knowlege of its sorting signals

1. in a tube:

   • radioactively labeled protein with and without signal sequence

   • incubated with target organelle

     —> Centrifugation: protein with signal sequence located in organelle and the free protein form two separate fractions

     —> protease degradation: organelle protects protein with signal sequence and free protein is degraded after adding detergent organelle is destroyed

2. tagging a protein with GFP (green fluorecent protein)

3. using genetics: Mutations in genes involved at different stages of the transport process

Post-tranlational sorting = peptide is released to cytosol (to be sorted after and is destined for nucleus, mitochondria or peroxisomes)

Co-translational sorting (secretory pathway) = the ribosomes with the nascent peptide are targeted to the ER (rough ER) and the peptide is sorted imediatly and destined for secretion from the cell or for the membranes [ER, Golgi, lysosomes]

1. signal sequence (AA sequence specific for a target organelle, mostly N-terminal sequence)

2. receptors for the signal sequence

3. translocation channels

4. Energy that drives unidirectional transfer across the membrane

Proteins (larger than 5 kDa) are imported through NPC (nuclear pore complex) and need to contain NLS (nuclear localization signal)

NPC has gel like meshwork so larger molecules cant just enter they need import receptors (importins) some NLS need adaptor proteins to bind, similar mechanism for export (exportins)

Ran GTPase: GTP binding by GEF in nucleus (Cargo unloading) and phosphate cleavage by GAP in cytosol (Cargo loading)

NF-AT (nuclear factor of activated T-Cells): acts as transcription factor when Ca2+ conc is high in the activated T-cell the nuclear import signal is dephosphorylated and an an inhibitory protein blocks the nuclear export signal NF-AT is transported into nucleus and activates gene expression, when Ca2+ conc is low in resting T-cell the inhibitory protein is removed so the export signal is active and the nuclear import signal is phosphorylated again the NF-AT gets transported out of nucleus

1. the mitochondrial signal sequence

2. receptors on TOM that bind signal sequence

3. transport over the double membrane via TOM (translocase outer membrane) and TIM (translocase inner membrane) complexes

4. energy (ATP) needed to remove chaperones at TOM and energy (membrane potential) needed for insertion into inner membrane and energy (ATP) to pull protein chain htrough TIM

If protein should be localized into membrane SAM (sorting and assembling machinery) complex does it in outer membrane and TIM 22 in inner membrane, into intermembrane space OXA

ATFS-1 is a transcription factor (in C elegans) that has both a mitochondrial import signal and a nuclear import signal (NLS) under normal conditions the mitochondrial import signal is dominant and ATFS-1 is transported over TOM-TIM into the mitochondrium and degraded by the LON protease.

Under mitochondrial stress the import over TOM complex is blocked resulting in transport into the nucleus via the nuclear localization signal, in the nucleus ATFS-1 activates the unfolded protein stress response (UPRmt)

Proteins Enter Peroxisomes from both the Cytosol and the Endoplasmic Reticulum

in particular in liver and kidney cells: ß-oxidation (break-down) of long chain and branched fatty acids

H2O2 detoxification

no DNA, no ribosomes!

signal sequence: SKL-Cterm, other N-term signals

import defects: Zellweger syndrome!

Proteins (with ER signal sequence detected by SRP [signal recognition particle] + receptor at membrane) enter the Endoplasmic Reticulum (trough translocator) while being synthesized (Ribosome become membrane bound) the signal sequence is cleaved by signal sequence peptidase —> CO-TRANSLATIONAL

Transmembrane proteins are already build through ER membrane depending on Start and Stop transfer sequences in the peptide chain (e.g. double pass TM proteins or 7 TM proteins like G-coupled receptors)

ER:

50% of cellular membranes

Functions: cotranslational import production of most lipids

all TM (transmembrane) proteins for most organelles

store for Ca2+ ions

protein distribution center (secretion, Golgi)

protein modifications (N-glycosylation) of proteins in ER lumen

misfolded ER proteins are ubiquitinated and degraded via proteasome after translocation to cytosol, UPRer is activated (IRE1, PERK, ATF6)

a. Nuclear Targeting Signal:

• Nature of Signal: The nuclear targeting signal, or nuclear localization signal (NLS), is typically a short stretch of amino acids rich in basic residues (lysine and arginine). Classical NLS sequences are commonly recognized by importins.

• Interpretation by the Cell: Importins are responsible for recognizing the NLS in the cytoplasm and facilitating the transport of the protein into the nucleus. The interaction between the NLS and importins is dynamic and regulated by Ran-GTP, allowing for the release of the cargo protein inside the nucleus.

b. Endoplasmic Reticulum (ER) Targeting Signal:

• Nature of Signal: Proteins destined for the endoplasmic reticulum often have a signal peptide at the N-terminus, which guides the ribosome-mRNA-polypeptide complex to the ER membrane. This signal peptide is typically cleaved upon translocation into the ER lumen.

• Interpretation by the Cell: The signal recognition particle (SRP) recognizes the signal peptide as it emerges from the ribosome. The SRP-ribosome complex is then targeted to the ER membrane, where the nascent polypeptide is translocated into the ER lumen through the translocon complex.

c. Mitochondrial Targeting Signal:

• Nature of Signal: Mitochondrial targeting signals vary but often include amphipathic helices or positively charged amino acids. These signals are located at the N-terminus or internally within the protein sequence.

• Interpretation by the Cell: Mitochondrial targeting signals are recognized by the translocase of the outer mitochondrial membrane (TOM) complex. Following recognition, the protein is translocated across the outer mitochondrial membrane. The translocase of the inner mitochondrial membrane (TIM) complex facilitates the translocation across the inner membrane.

d. Extracellular Space Targeting Signal:

• Nature of Signal: Proteins destined for secretion or the extracellular space often contain a signal peptide at the N-terminus. This signal is usually cleaved after translocation across the endoplasmic reticulum membrane.

• Interpretation by the Cell: Similar to proteins targeted to the ER, the signal recognition particle (SRP) recognizes the signal peptide during translation. The SRP-ribosome complex is targeted to the ER membrane, and the nascent polypeptide is translocated into the ER lumen. Following processing, the protein is transported through the Golgi apparatus and eventually secreted to the extracellular space.

a. Localization Signals:

• The mitochondrial localization signal in ATFS-1 is expected to be localized within the amino acid sequence near the N-terminus

• Principle Features of Localization Signals:

  • Mitochondrial Localization Signal: Typically amphipathic helices, positively charged amino acids, or specific motifs that are recognized by the mitochondrial import machinery.

  • Nuclear Localization Signal: Enriched in basic amino acids, especially lysine and arginine residues. Recognized by nuclear import receptors (importins).

b. Engineered ATFS-1 Variants:

• if nucleus signal is missing it would not change in default mode because mito signal is dominant but in case of stress response it would not be located into nucleus

• if mito signal is removed it would be localized into nucleus always triggering the stress response

c. Masking Nuclear Targeting Signal:

• Mechanism: The cell can use post-translational modifications (phosphorylation) or protein-binding partners to mask the nuclear localization signal (NLS) temporarily.

• Examples: Proteins like importins, which typically recognize NLS, can be bound to other proteins or undergo modifications, preventing them from interacting with the NLS. Another example is NF-AT in T-cell activation, where the import signal is blocked by phosphorylation and the export signal can be blocked by calcineurin (when import needed —> T-cell activation with high Ca2+)

d. Mitochondrial Import and LONP-1 Interaction:

• TOM-TIM import transports ATFS-1 to mito matrix where LONP-1 protease degrades it, the import machinery contributes by transporting it over both outer and inner membrane and by cleaving the mitos targeting signal by MPP (mitochondrial processing peptidase), possibly during the import specific regions for degradation (cleavage sites) are exposed for the LON protease.

e. Impact of Electron Transport Chain (ETC) Efficacy:

• Scenario: Lowering the efficacy of the electron transport chain typically leads to mitochondrial stress —> TOM complex is inhibited

• Consequence for ATFS-1: In the case of mitochondrial stress, ATFS-1 is not efficiently imported into the mitochondria. Instead, it accumulates in the cytoplasm. This allows ATFS-1 to translocate to the nucleus, where it induces the expression of nuclear-encoded mitochondrial stress response genes.