Endoplasmic Reticulum & Protein Targeting

Rough Endoplasmic Reticulum (RER) – Context & Importance

• RER = membranous network covered in ribosomes ➔ primary site for synthesis of:
  • All membrane-embedded proteins (carriers, channels, pumps, receptors)
  • Proteins that will be secreted or reside inside endomembrane organelles
• Smooth ER, Golgi, etc. will be discussed later; today’s focus = how ribosomes get onto RER and what happens next.

Ribosome Association with RER

• Electron micrographs show only complete cytosolic ribosomes bound to RER; you never see isolated sub-units.
  • Full eukaryotic ribosome = $80\,S = 60\,S + 40\,S$
  • Conclusion ➔ sub-units must first assemble in cytosol, begin translation, then dock as a unit.
• Not every ribosome can bind; only those translating a specific mRNA class do so.

Signal/Leader Sequence (“Hydrophobic Leader”)

• Located within the first ≈20 codons of certain mRNAs.
• Translation of those codons ➔ N-terminal ~20 amino-acid peptide that is:
  • Highly hydrophobic (avoids aqueous cytosol)
  • Called the leader sequence / signal peptide
• Key roles:
  1. Targets ribosome–mRNA–nascent-peptide complex to RER membrane.
  2. Pauses elongation until docking occurs (prevents incomplete protein from floating freely).

“Decision Point” – With vs. Without a Leader

• WITH leader (signal peptide)
  • Ribosome pauses ➔ docks to RER via signal-recognition pathway (details implicit).
  • Translation re-starts; growing polypeptide is threaded into/through RER membrane or lumen.
  • Leader peptide is cleaved off (by a signal peptidase) once inside.
• WITHOUT leader
  • Translation proceeds entirely in cytoplasm.
  • Protein folds to its tertiary structure locally and functions in cytosol, nucleus, mitochondria, etc.
• Central concept: leader presence = RER destination; absence = cytosolic destination.

Classes of Proteins Synthesised on RER

• Integral membrane proteins (all types of transport/channel proteins previously studied).
• Secretory proteins destined for extracellular export.
• Soluble proteins meant for ER, Golgi, lysosomal lumen, or extracellular matrix.
• Contrast: Metabolic enzymes (e.g., glycolytic enzymes) stay cytosolic; they lack leader sequences.

Post-Translational Modifications (PTMs) in/at the RER

• Distinct from post-transcriptional mRNA mods (capping, poly-A, intron removal).
• RER-linked PTMs covered today:
  1. Leader removal (proteolytic clipping).
  2. Glycosylation – covalent attachment of custom-built oligosaccharide to nascent protein.

Dolichol & Oligosaccharide Assembly

• Dolichol phosphate = enormous transmembrane lipid spanning both monolayers of the RER membrane; acts as a scaffold for sugar assembly.
• Steps (overview):
  1. On cytosolic face, monosaccharides are sequentially added → build a defined oligosaccharide.
  2. Core sugar composition heavily features:
  • $\text{N-acetylglucosamine (GlcNAc)}$ – the most common “base” unit.
  • Mannose and glucose – also abundant, though other sugars can appear.
  1. Fully assembled oligosaccharide is positioned for transfer to the target polypeptide.

N-Linked Glycosylation (Primary RER Sugar Attachment)

• Attachment site: side-chain amino nitrogen ((\text{–NH}_2)) of a specific asparagine (Asn) residue.
• Consensus sequence within the protein:
  • $\text{Asn – X – Ser/Thr}$
    ((X) = any amino acid except proline; ensures correct local folding/exposure.)
• Because sugar is linked via the nitrogen atom, process is termed N-linked glycosylation.
• Result = glycoprotein – a protein now bearing an oligosaccharide essential for proper folding, stability, quality control, and eventual targeting.

Fate After Initial RER Processing

• Newly formed glycoprotein remains in RER (lumen or membrane) until further sorting.
• Upcoming steps (to be detailed in later lectures):
  • Quality-control chaperones, folding cycles, vesicular transport to Golgi, trimming/extension of glycans, etc.

Connections & Implications

• Central Dogma linkage: DNA → (pre-mRNA w/ introns) → mRNA (post-transcriptional mods) → Protein (translation) → Protein + PTMs (leader removal, glycosylation) → Functional product.
• Hydrophobic leader sequences exemplify how primary sequence encodes targeting information, reinforcing the gene ➔ function theme.
• Clinical relevance: Mutations that disrupt leader sequences, consensus Asn sites, or dolichol-linked glycosylation enzymes cause congenital disorders of glycosylation (CDGs) and ER-stress diseases.
• Biotechnological angle: Recombinant production of secreted antibodies, hormones, etc., requires eukaryotic expression systems precisely because they possess RER-based glycosylation machinery absent in bacteria.

• Next lecture will shift focus to the smooth ER and additional processing/trafficking steps.