Gene Expression Regulation: Post-Translational Control and Protein Trafficking

Overview of Post-Translational Control

• Definition and Context: Post-translational control represents the final stages of gene expression regulation within the central dogma of molecular biology.
• Key Processes Included:
  • Protein trafficking: Directing proteins to their necessary cellular locations.
  • Protein folding and processing: Ensuring the three-dimensional structure is functional and applying necessary covalent modifications (post-translational modifications or PTMs).
  • Protein recycling: Systematically degrading proteins that are no longer needed or are misfolded.

Protein Trafficking and Sorting

• The Problem of Sorting: As proteins are synthesized, the cell must accurately sort and direct them to their final destinations to ensure cellular function and compartmentalization.
• Instructional "Tags": Trafficking is mediated by specific molecular signals or tags within the protein structure. These include:
  • Short Amino Acid (AA) sequences.
  • Oligosaccharides (sugar chains).
  • Hydrophobic motifs.
• Major Synthesis Site: Most proteins are initially synthesized on ribosomes within the cytoplasm.
• Primary Destinations: Destinations include the cytosol, mitochondria, chloroplasts, peroxisomes, nucleus, endoplasmic reticulum (ER), Golgi apparatus, or secretion outside the cell.

Post-Translational vs. Co-Translational Import

• Post-Translational Import:
  • Targets: Cytosol, mitochondria, chloroplasts, peroxisomes, and the nucleus.
  • Mechanism: Translation occurs entirely on "free" ribosomes in the cytosol.
  • If the protein is destined for the cytosol, it remains in the cytoplasm once translation is complete.
  • If destined for an organelle, various recognition mechanisms identify the signal and traffic the completed protein to the destination.
• Co-Translational Import:
  • Targets: Proteins destined for the endomembrane system (ER, Golgi, lysosomes), integral membrane proteins, or proteins destined for export (secretion).
  • Mechanism: Translation begins on a ribosome but is soon directed to the ER membrane. The polypeptide chain is transferred across the ER membrane as translation is occurring.

ER Signal Peptides and the Signal Recognition Particle (SRP)

• Preproteins: Proteins destined for co-translational insertion initially contain an N-terminal sequence targeting them to the ER.
• ER Signal Sequence Characteristics:
  • Located at the N-terminus.
  • Typically comprises $15-30$ amino acids.
  • Directs the entire ensemble (mRNA, ribosome, and growing polypeptide) to the ER.
  • This sequence is typically removed once the protein reaches the ER.
  • Experimental evidence: Adding an ER signal sequence to a protein that is normally non-ER-destined will cause that protein to be imported into the ER.
• Signal Recognition Particle (SRP) Structure: A complex consisting of $6$ proteins and one RNA molecule approximately $300$ nucleotides ($nt$) in length.
• Mechanism of Co-translational Import:
  1. The SRP binds to the ER signal sequence as it emerges from the ribosome and temporarily blocks further translation.
  2. The SRP-ribosome complex binds to the SRP receptor located on the ER membrane; the ribosome docks on a membrane channel called a translocon (specifically the Sec61 complex).
  3. $GTP$ binds to both the SRP and the SRP receptor. This interaction opens the translocon pore, and the polypeptide is inserted.
  4. Both $GTP$ molecules are hydrolyzed to $GDP$, which triggers the release of the SRP.
  5. As the polypeptide elongates and moves into the ER lumen, a signal peptidase cleaves the signal sequence.
  6. The completed polypeptide is released into the ER lumen, the ribosome dissociates, and the translocon pore closes.

Trafficking Integral Membrane Proteins

• Type I Transmembrane Proteins:
  • Single-pass proteins with the $COO^-$ (carboxyl) end located in the cytosol.
  • Trafficked co-translationally using an N-terminal ER signal.
  • Contain a hydrophobic $\alpha$-helix domain that acts as a "stop-transfer" signal. This sequence stops the translocation of the polypeptide into the ER lumen, causing the remaining portion of the protein to stay on the cytosolic side.
• Type II Transmembrane Proteins:
  • Single-pass proteins with the $NH_3^+$ (amino) end located in the cytosol.
  • They lack the N-terminal ER signal.
  • Possess an internal hydrophobic $\alpha$-helix domain that serves as a "start-transfer" signal.
  • The SRP binds to this internal signal and targets the complex to the ER membrane.
• Multi-pass Transmembrane Proteins:
  • Utilize multiple start-transfer and stop-transfer signals to "thread" the protein back and forth through the ER membrane.
  • The process alternates between patterns similar to Type I and Type II signals until all segments are properly inserted.

Protein Folding and the Unfolded Protein Response (UPR)

• Necessity of Folding: Proteins must achieve their correct three-dimensional shape to be functional.
• Molecular Chaperones: Specialized proteins that facilitate folding within the ER.
  • Hsp70: Acts in the ER to assist new proteins. It loosely binds the polypeptide and uses energy from $ATP$ hydrolysis to bind tightly. Repeated cycles of $ATP$ hydrolysis assist in folding the polypeptide.
  • Hsp60 (Chaperonin): Assists partially folded or misfolded proteins. In prokaryotes, this is known as the GroEL/GroES complex. The polypeptide enters a subunit; $ATP$ hydrolysis induces a conformational change that creates a hydrophilic environment favorable for folding. Once folded, the protein is released.
• Unfolded Protein Response (UPR): A cellular stress response triggered by repeated failures in protein folding. The UPR shuts down cap-dependent translation to allow the cell to "catch up" and prevent the accumulation of toxic misfolded proteins.

Post-Translational Processing

• N-terminal Processing: In both prokaryotes and eukaryotes, the initial N-terminal Methionine is almost always removed after translation.
• Assembly and Cleavage:
  • Assembly into tertiary and quaternary structures often occurs post-translationally.
  • Specific amino acid sequences may be cleaved to activate a protein.
• Example: Insulin Processing:
  1. Preproinsulin: The initial inactive precursor.
  2. Removal of N-terminal "pre" amino acids converts preproinsulin to proinsulin.
  3. Disulfide bonds form between subunits, beginning the formation of the 3D shape.
  4. The "pro" amino acids connecting the subunits are removed, resulting in mature insulin with its final 3D structure.

Post-Translational Modifications (PTMs) and Regulation

• Function: PTMs are rapid, dynamic mechanisms to toggle proteins between active and inactive states.
• Common PTMs:
  • Methylation.
  • Phosphorylation (often involves kinases and phosphatases).
  • Acetylation.

Protein Recycling Mechanisms

• The Proteasome Pathway:
  • Ubiquitin Tags: Small proteins that act as "garbage tags" to mark proteins for destruction.
  • E1 (Ubiquitin-activating enzyme): Prepares ubiquitin for attachment.
  • E2 (Ubiquitin-conjugating enzyme): Attaches the ubiquitin molecule to the target protein.
  • E3 (Substrate recognition protein): Provides specificity by determining which specific protein should be ubiquitinated.
  • The Proteasome: A large protein complex in the cytoplasm that recognizes ubiquitinated proteins and degrades them into small peptides.
• The Lysosome (Microautophagy):
  • A membrane-bound organelle that acts as a digestive center.
  • Generally involves non-selective recycling.
  • Proteins are taken up by the infolding of the lysosomal membrane and digested into individual amino acids.

Information Flow Discrepancies in the Central Dogma

• Non-Equivalence: Regulation at multiple levels means that the abundance of components does not scale linearly:
  • $\text{DNA abundance} \neq \text{mRNA abundance}$
  • $\text{mRNA abundance} \neq \text{protein abundance}$
• Example Case Study: If two genes have identical mRNA levels but Protein A is present at $20\times$ the level of Protein B, potential causes include:
  • Differences in translation efficiencies (e.g., presence of a uORF or upstream Open Reading Frame in Protein B which inhibits translation).
  • Differences in mRNA stability.
  • Differences in the rate of protein degradation via ubiquitination.
• Temporal Dynamics of Control:
  • Early Control (DNA/Transcription): Takes longer to implement but results in more long-term effects.
  • Late Control (Post-translation): Is rapid and allows for short-term, dynamic responses to environmental changes.

Questions & Discussion

• Q1: Recall, where are most proteins made?
  • Response: Most proteins are made on ribosomes in the cytosol.
• Q2: What are the main destinations for proteins?
  • Response: Destinations include the cytosol, mitochondria, chloroplasts, peroxisomes, nucleus, ER, Golgi, and extracellular space.
• Q3: What are some common features of AAs in a signal peptide?
  • Response: They are typically $15-30$ amino acids long and located at the N-terminus.
• Q7: Why would we include several processing steps to make mature insulin instead of immediately translating the mature protein?
  • Response: Processing steps allow for precise spatial and temporal control of activity, ensuring insulin is only active when and where it is needed.
• Q10/Q11: Scenario of identical mRNA levels but $20\times$ difference in protein levels.
  • Factors: Translation efficiency, mRNA stability, and ubiquitination rates.
  • Mechanism: A uORF (upstream Open Reading Frame) typically decreases the translation of the main coding sequence, explaining lower protein levels despite equal mRNA levels.