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Post-translational modification (PTM): overview

• PTMs occur after polypeptide synthesis and can modify protein function, localization, interactions, degradation, and cell death regulation.
• Two broad classes by reversibility:
  • Reversible PTMs: examples include phosphorylation (adding phosphate groups), glycosylation (adding sugars), and ubiquitination (adding ubiquitin tags).
  • Irreversible PTMs: exemplified by proteolysis (cleavage of peptide bonds). A classic example discussed is pepsinogen → pepsin in the stomach, where an inactive precursor is activated to digest proteins.
• Relevance of PTMs:
  • Critical for trafficking/transport of proteins, cell–cell interactions, and regulation of protein degradation.
  • Abnormal PTMs can affect multiple organ systems and contribute to disease.
• Not all PTMs are listed exhaustively here; focus for this course includes the four highlighted processes: proteolysis, ubiquitination, glycosylation, and the conceptual role of PTMs in trafficking and function.

Key concepts about PTMs and organelles

• Proteolysis (irreversible): a protease removes a portion of a protein to activate or mature it (e.g., pepsinogen → pepsin).
• Ubiquitination (reversible in the broader sense of dynamic tagging): ubiquitin tags mark proteins for degradation via the proteasome.
• Glycosylation (reversible in some contexts; often considered permanent for a given protein once set): attachment of sugar moieties can affect folding, stability, and trafficking.
• PTMs influence: protein trafficking, intercellular interactions, selective degradation, and apoptosis.

Endoplasmic reticulum (ER): two types and their roles

• Rough ER (RER): has ribosomes on its surface; primary site for synthesis of secretory and membrane proteins; central to proteostasis (protein homeostasis).
• Smooth ER (SER): lacks ribosomes; functions include detoxification, lipid synthesis, and calcium storage; especially prominent in detoxification organs like the liver.
• Sarcoplasmic reticulum: a specialized form of SER in muscle cells; stores calcium to regulate contraction/relaxation.
• Proteostasis: two components
  • Protein synthesis (biosynthesis of new proteins)
  • Protein quality control (proper folding, maturation, stabilization, and trafficking)
  • In formula: $\text{Proteostasis} = \text{protein synthesis} + \text{protein quality}.$
• Protein maturation in ER: after synthesis, proteins fold, may undergo PTMs, and are trafficked to their destinations (cell membrane, secretion, or extracellular space).

Co-translational translocation into the ER (how proteins enter the ER lumen or membrane)

• Objective: deliver cytosolic proteins into the ER during translation for ER-targeted or membrane proteins.
• Key players:
  • Signal peptide: a hydrophobic sequence at the N-terminus of the nascent polypeptide that targets the ribosome-nascent chain to the ER.
  • Signal recognition particle (SRP): binds the signal peptide and pauses translation; directs ribosome-nascent chain to ER.
  • SRP receptor: located on the ER membrane; anchors SRP and guides the complex to the translocon.
  • Translocon (Sec61 complex): a protein-conducting channel in the ER membrane; forms a conduit for translocation into the ER lumen or integration into the membrane.
  • Signal peptidase: cleaves the signal peptide after its job is done, and the nascent chain continues into the ER lumen or integrates into the membrane.
  • Lateral gate: a side entry in the translocon that allows hydrophobic segments to integrate into the lipid bilayer (for transmembrane domains).
  • BiP and ER chaperones (e.g., BiP/VIP): assist in folding once inside the ER lumen.
• Step-by-step (simplified):
  1) The SRP binds the signal sequence on the nascent chain and directs the ribosome–nascent chain–RNA complex to the SRP receptor on the ER.
  2) The ribosome–nascent chain complex is loaded onto the Sec61 translocon.
  3) The signal sequence engages the signal recognition site on the translocon; translation continues, pushing the polypeptide through the channel into the ER lumen.
  4) The hydrophobic signal peptide is cleaved by the signal peptidase and degraded; the remaining polypeptide enters the ER lumen if hydrophilic or enters the lipid bilayer via the lateral gate if hydrophobic segments become transmembrane domains.
  5) Inside the ER lumen, resident chaperones (e.g., BiP) bind to assist folding and early maturation.
• Important caveat discussed in class notes:
  • The phrase “polypeptide loop” refers to the growing hydrophobic region within the translocon; ensure correct terminology: the hydrophobic region is the signal sequence/polypeptide loop that interacts with the translocon and drives translocation.
• Visual metaphor used: the translocon is like a barrel with two main openings (top and bottom) and a lateral/gate entry for inserting transmembrane domains.

Transmembrane proteins and multi-pass topology

• Multipass transmembrane proteins: contain two or more hydrophobic transmembrane domains embedded across the ER membrane.
• Basic mechanism:
  • The start transfer cue is the ER signal sequence (first transmembrane domain or a signal anchor sequence).
  • The lateral gate opens when a hydrophobic region is encountered and inserts that domain into the lipid bilayer.
  • Translation then resumes, producing cytosolic domains until the next hydrophobic region is encountered.
  • The cycle repeats for additional transmembrane domains until the entire protein is embedded with the correct topology.
• Orientation rules:
  • Proteins with an odd number of transmembrane domains have their N- and C-termini on opposite sides of the membrane.
  • Proteins with an even number of transmembrane domains have their N- and C-termini on the same side.
• Practical outcomes:
  • The first transmembrane domain is inserted via SRP–translocon mechanism.
  • Hydrophobic segments drive membrane insertion via the lateral gate; hydrophilic regions tend to reside in the lumen or cytosol as directed by topology.
• Summary concept: synthesis occurs inside the translocon, and whether a segment ends up in the membrane or lumen depends on its hydrophobicity and sequence context during translation.

Post-entry ER folding, quality control, and vesicular trafficking

• ER chaperones promote proper folding and initial PTMs (e.g., early glycosylation, disulfide bond formation, oligomerization).
• Properly folded proteins are packaged into vesicles for transport to their destinations:
  • Cell membrane (as transmembrane proteins) or
  • Secretion into extracellular space (secreted proteins like hormones, antibodies).
• ER quality control and misfolded proteins:
  • Misfolded proteins are recognized by chaperones and targeted for degradation via ER-associated degradation (ERAD).
  • ERAD uses the ubiquitin–proteasome system (UPS): misfolded protein recognition → ubiquitination → retrotranslocation to the cytosol → proteasomal degradation.
  • Lysosomes also play a role for degraded proteins via autophagic pathways (see below).
• Precise pathway for misfolded proteins (ERAD):
  1) Recognition by chaperones (misfolded markers shown as red stars).
  2) Ubiquitination of the misfolded polypeptides (Ub tagging on the substrate).
  3) Retrotranslocation (export from the ER back into the cytosol).
  4) Degradation by the proteasome (protein breakdown and amino acid recycling).
• UPR: Unfolded Protein Response (ER stress response)
  • Triggered by accumulation of misfolded proteins in the ER due to various stresses (lipid/protein/glucose imbalances).
  • Three-step program:
    1) Attenuate general protein synthesis to reduce the load of new proteins entering the ER.
    2) Increase production of ER chaperones to assist with proper folding.
    3) If homeostasis cannot be restored, promote autophagy to degrade misfolded components via autophagosomes and lysosomes.
• Autophagy details:
  • Autophagosomes engulf cellular components; fuse with lysosomes where degradation occurs.
  • Recycled components re-enter cellular metabolism; non-recyclable parts are degraded.

Golgi apparatus: sorting and post-translational modifications

• Structure: stack of flattened, membrane-bound sacs called cisternae.
  • Cis face: near the nucleus/ER; receives vesicles from the ER.
  • Trans face: near the cell membrane; vesicles exit toward destinations.
• Functional analogy: the Golgi functions like a post office, sorting and labeling packages for their destinations.
• Modifications in transit: additional post-translational modifications occur as proteins traverse the Golgi (e.g., further glycosylation, phosphorylation).
• Destination vs destiny (three destinations):
  • Destination: where the protein is sent next (e.g., lysosome, plasma membrane, extracellular space).
  • Destiny: the ultimate fate after reaching the destination (functional outcome such as a receptor on the cell surface or secreted hormone).
• Three major fates of Golgi-processed proteins:
  1) Misfolded proteins directed to lysosomes for degradation (lysosomal degradation).
  2) Transmembrane proteins delivered to the plasma membrane (integrated into the membrane as single-pass or multi-pass proteins).
  3) Secreted proteins released into the extracellular space (e.g., hormones, antibodies).
• Visualization tip used in lecture:
  • Vesicles from the ER enter the Golgi at the cis phase, move through the cisternae, and exit at the trans phase to their destinations.

Recap: organelles and their roles in protein handling

• Nucleus:
  • DNA replication occurs here; transcription of RNA; RNA editing occurs in the nucleus as part of gene expression regulation.
• Endoplasmic reticulum (ER):
  • Co-translational translocation delivers certain proteins into the ER lumen or membrane during translation.
  • ER functions include protein synthesis, folding, maturation, PTMs (e.g., glycosylation, disulfide bonds), and quality control via ERAD and the UPR.
• Golgi apparatus:
  • Further processing and sorting of proteins from the ER.
  • Destination and destiny labeling determine whether proteins go to lysosomes, the plasma membrane, or extracellular space.

Quick poll note from lecture (conceptual check)

• Question: The presence of a signal peptide in a protein sequence indicates that it will likely be processed in which compartment?
  • Options discussed: rough ER, Golgi apparatus, cytoplasm, or all of these.
  • The lecturer prompted the class to answer with cytoplasm; the rationale given was that the signal peptide is hydrophobic and not stable in the aqueous cytosol, implying trafficking toward the ER.
  • The intended mechanistic concept is that signal peptides direct ribosome-nascent chain complexes to the ER for co-translational translocation; therefore, processing is expected to occur primarily in the rough ER (and further in the Golgi for many proteins, and beyond if secreted).
• Practical takeaway: a signal peptide targets to the ER for co-translational translocation; cytoplasm is not the primary site of processing for proteins bearing a signal peptide.

Final recap: three major organelles and their protein-handling roles

• Nucleus: DNA replication; transcription and RNA processing/editing.
• ER: site of protein synthesis for secretory/molded proteins; co-translational translocation; folding and initial PTMs; quality control via ERAD and UPR.
• Golgi: sorting and final modifications; determines ultimate destination (lysosome, plasma membrane, secretion).

Formulas and key definitions (LaTeX)

• Proteostasis relationship:
  $\text{Proteostasis} = \text{protein synthesis} + \text{protein quality}$
• Ubiquitin–proteasome system (UPS): components include ubiquitination tagging and proteasomal degradation; ERAD is the ER-specific degradation pathway feeding substrates to the UPS.
• Key terms:
  • SRP: Signal Recognition Particle
  • SRP receptor: receptor on the ER membrane
  • Sec61 translocon: ER membrane channel for polypeptide entry
  • Signal peptide: hydrophobic N-terminal sequence targeting ER
  • Lateral gate: side entrance of the translocon for membrane integration
  • ERAD: ER-associated degradation
  • UPR: Unfolded Protein Response
  • Autophagy: self-eating process delivering components to lysosomes for degradation

Notes on video content and mechanism (takeaways)

• The ER translocation process can be visualized as a coordinated handoff: SRP binds signal peptide, SRP–receptor guides ribosome to translocon, translation continues, signal peptide is cleaved, and the growing polypeptide enters the ER lumen or integrates into the membrane via the lateral gate.
• Chaperones (e.g., BiP) in the ER lumen assist folding and maturation; misfolded proteins trigger ERAD and/or UPR to restore homeostasis.
• The Golgi serves as a processing and sorting hub, tagging proteins to ensure appropriate destination, with three principal fates described: lysosomal degradation, plasma membrane incorporation, or secretion.
• The presence of a signal peptide is a marker for ER targeting; cytoplasmic processing is unlikely for such proteins, and the signal peptide signals eventual ER/Golgi handling rather than cytoplasmic processing.