Protein Production and Secretion

So far we have studied each organelle in isolation, but a key theme at A-Level is that organelles work together as an integrated system. Nowhere is this more elegantly demonstrated than in the production and secretion of proteins. OCR 2.1.1 (g) asks you to explain the interrelationship between the organelles involved — from the synthesis of ribosomal RNA in the nucleolus through to the release of a mature protein at the plasma membrane by exocytosis.

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An Overview of the Journey

A secreted protein (for example, the digestive enzyme amylase made by pancreatic cells, or the hormone insulin made by β-cells of the pancreas) passes through the following steps:

1. Gene transcription in the nucleus produces mRNA.
2. rRNA synthesis in the nucleolus assembles ribosomal subunits.
3. Both mRNA and ribosomal subunits exit the nucleus through nuclear pores.
4. The small subunit and large subunit combine with mRNA in the cytoplasm to form a functional 80S ribosome.
5. The ribosome begins translation. A signal sequence at the start of the polypeptide directs it to the rough endoplasmic reticulum (RER).
6. The polypeptide enters the RER lumen, where it is folded and modified (e.g., glycosylated).
7. Transport vesicles pinch off from the RER carrying the new protein.
8. Vesicles move via the cytoskeleton and fuse with the cis face of the Golgi apparatus.
9. The protein is modified further as it passes through the Golgi cisternae.
10. At the trans face, secretory vesicles bud off carrying the mature protein.
11. Vesicles travel to the plasma membrane along cytoskeletal tracks.
12. Vesicles fuse with the plasma membrane, releasing the protein outside the cell in a process called exocytosis.

Throughout this process, mitochondria supply the ATP needed for transcription, translation, vesicle movement, modification, and exocytosis.

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Step-by-Step Detail

1. The Nucleus and Transcription

• In the nucleus, an enzyme called RNA polymerase binds to a specific gene (e.g., the insulin gene).
• It unwinds the DNA double helix and uses one strand as a template.
• Free RNA nucleotides base-pair with the DNA template (A–U, C–G) to form a complementary strand of pre-mRNA.
• The pre-mRNA is processed: a 5' cap is added, introns are spliced out, exons are joined, and a poly-A tail is added. The result is mature mRNA.

2. The Nucleolus and rRNA Synthesis

• Within the nucleus, the nucleolus contains special chromosomal regions that carry many copies of the rRNA genes.
• rRNA is transcribed here by RNA polymerase I.
• Ribosomal proteins, imported from the cytoplasm, are assembled with rRNA to form the 60S large ribosomal subunit and the 40S small ribosomal subunit.
• Both subunits are exported separately through nuclear pores to the cytoplasm.

3. Exit Through Nuclear Pores

• Both mRNA and the large/small ribosomal subunits pass through nuclear pore complexes in the nuclear envelope.
• These are large protein channels, about 100 nm wide, that selectively transport macromolecules between nucleus and cytoplasm.

4. Ribosome Assembly and Translation

• In the cytoplasm, a large (60S) subunit, a small (40S) subunit, and an mRNA molecule assemble to form a functional 80S ribosome.
• Translation begins: tRNAs deliver amino acids matching each mRNA codon, and the ribosome catalyses peptide bond formation, growing a polypeptide chain.

5. Targeting to the RER

• Proteins destined for secretion begin their synthesis on free ribosomes, but the first ~20 amino acids of the growing polypeptide form a signal sequence.
• A signal recognition particle (SRP) in the cytoplasm binds to the signal sequence and halts translation.
• The SRP–ribosome complex docks onto an SRP receptor on the RER membrane. A translocon (membrane channel) opens, and translation resumes, feeding the growing polypeptide into the RER lumen.
• Once the protein has entered, the signal sequence is cleaved by a signal peptidase.

6. Folding, Modification and Quality Control in the RER

• Inside the lumen of the RER, chaperone proteins help the polypeptide fold correctly.
• Disulfide bridges may form between cysteine residues.
• Glycosylation — sugar chains are added to specific amino acids (typically asparagine residues), converting the protein into a glycoprotein.
• Misfolded proteins are detected by quality-control systems and retro-translocated back to the cytoplasm for destruction by the proteasome.

7. Transport Vesicles from RER to Golgi

• Correctly folded proteins are packaged into transport vesicles that bud off from the RER.
• These vesicles are coated with coat proteins (COPII in this direction) that drive membrane curvature.
• The vesicles move along microtubules, powered by kinesin and dynein motors, to the cis face of the Golgi apparatus.

8. Through the Golgi Apparatus

• Transport vesicles fuse with the cis-Golgi and release their contents into the lumen.
• The protein passes through the stacked cisternae (cis → medial → trans), undergoing progressive modifications:
  • Further trimming and extension of sugar chains.
  • Addition of new sugar groups specific to the final destination (e.g., mannose-6-phosphate for proteins destined for lysosomes).
  • Proteolytic cleavage — for example, the hormone proinsulin is cleaved to form mature insulin plus a discarded C-peptide.
  • Sulphation, phosphorylation, and other post-translational modifications.

9. Sorting at the Trans-Golgi Network

• At the trans face, proteins are sorted into different types of vesicle:
  • Secretory vesicles → secretion outside the cell.
  • Lysosomes → intracellular digestion.
  • Membrane vesicles → new plasma membrane proteins.