Wk 5.2 Biosynthetic Secretory Pathway

Biosynthetic Secretory Pathway Overview
- Focus on the transport of secretory and organelle proteins across the endoplasmic reticulum (ER) membrane.
- Understand the biosynthetic secretory pathway processes, including sorting, compartments traversed, and post-Golgi sorting/maturation.

Ribosome Binding Process (SRP Cycle)
1. Signal Sequence: Part of the growing peptide recognised by the Signal Recognition Particle (SRP).
2. SRP Binding:
  - SRP binds to the signal peptide, causing translation to pause.
3. Targeting:
  - SRP connects to the SRP receptor in the rough ER membrane.
4. Translation Resumption:
  - Translation resumes, and the polypeptide is translocated into the ER lumen.

Signals and Receptors
- Proteins contain sorting signals (15-60 amino acids, often at N-terminus) that direct them to the correct cellular location.
- Cleaved by signal peptidase following delivery
- Signals may include internal sequences that form specific configurations (e.g., nuclear localisation signals). aka. signal patch
Protein Characteristics
- Hydrophobicity/charge of the sequence often determines sorting, rather than exact amino acid composition.
- Necessary and sufficient for targeting — switch signals, redirect protein

SRP Mechanism:
- Signal Recognition Particle (SRP-protein complex) directs proteins to the ER membrane based on the presence of an ER signal sequence.
- SRP binds to large ribosomal subunit (signal sequence binding pocket binds near nascent chain exit site on ribosome)
Protein Translocators (Sec61 Complex):
- Forms a water-filled pore in the ER membrane allowing polypeptide chains to pass through.
- Can open in two ways: by moving a “plug” out of way or opening sideways via a seam
  - Signal sequence is released into the membrane after opening of seam

Folding and Assembly:
- Correct protein folding occurs in the ER; proteins assemble into multimeric forms.
- Disulphide bonds formed in the ER stabilise protein structure.
  - Oxidative linkage of sulphydryl (-SH) groups — a spontaneous reaction (sufficient oxidant)
  - Proper pairing Cys is essential for protein structure/ activity
  - Also catalysed by protein disulphide isomerase (PDI)
  - Form specific order » small domains first, then distant segments
Glycosylation:
- Most membrane and secretory proteins undergo glycosylation in the ER and Golgi apparatus, adding oligosaccharides.
- Important for protein stability, targeting, and function.
Special glycosylation » GPI anchor
- Some proteins acquire a covalently attached glycosyphosphatidyl-inositol (GPI) anchor
  - Added to C-terminus
  - Marks proteins for membrane insertion
  - These proteins are only attached by GPI anchor
  - Allows for soluble extracellular release by cells

Gated transport
- Between nucleus and cytoplasm
- Nuclear pore complexed (nuclear pore envelope)
- Selective ‘gate’, not free diffusion
Transmembrane transport
- Protein translocators move proteins from cytoplasm to topologically distinct areas (e.g. mitochondria, ER)
- Unfolded to across
Vesicular transport
- Membrane enclosed transport intermediates (vesicles) move proteins between compartments
- Loaded in lumen, bud from membrane, fuse to second compartment

After transport vesicles have budded they fuse – vesicular tubular clusters
Move along microtubules to carry proteins from ER to Golgi apparatus (bud off their own transport vesicles which carry escaped resident proteins and proteins involved in budding)
Direction depends on coating
- COPII » ER to Golgi
- COPI » retrieval transport

Structure:
- Comprises a series of flattened vesicles with cis, medial, and trans regions.
- Proteins arrive from the ER and continue to undergo post-translational modifications.
- Further modification of oligosaccharide chains occurs during transport through Golgi
- Each cisternae have different enzymes which modify proteins differently
Oligosaccharide Processing in Golgi Apparatus:
- Resident proteins are all membrane bound
- Finishes processing of oligosaccharides that started in ER

Transport Models:
- Vesicular Transport Model:
  - Static structure where proteins move through cisternae carried by transport vesicle (cis » trans)
- Cisternal Maturation Model:
  - Dynamic model describing how cisternae move,
  - Vesicular tubular cluster fuse to form cis (Golgi network) » medial (cisterna) » trans (cisterna)

Final Sorting in Trans-Golgi:
- Sorting signals determine the protein's final destination (e.g., lysosome or secretion).
- Enzymes that function in Golgi (e.g. glycosyltransferases) are retained by inserting in membrane (specific membrane spanning domain)
- Secretory proteins are then packaged into transport vesicles and taken to the cell surface
Secretion Proteins » To Surface:
- Proteins can be continuously secreted or regulated in response to stimuli (e.g., insulin and blood glucose).
- Some proteins can undergo cleavage in the vesicles

Proproteins require additional processing to become active (‘mature’) which occurs in secretory vesicles
Secreted proteins are cleaved by proteases prohormone convertase 2 (PC2) and 3 (PC3) and furin
Example: Proinsulin » folds » cleaved » insulin

Key Steps:
1. Protein synthesis occurs on ribosomes (both free and bound).
2. Proteins translocated across the ER and undergo modifications (disulphide bonds, glycosylation).
3. Move to Golgi for further processing.
4. Proteins reach the trans Golgi and are secreted or sent to specific organelles.