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Cell Biology Unit 3: Protein Sorting and the Endomembrane System

This unit follows proteins from the ribosome to their final address and explains how vesicles move cargo between the ER, Golgi, lysosomes, plasma membrane and the outside of the cell.

Covers: Signal sequences and SRP-dependent ER targeting, Membrane protein topology, ER folding, glycosylation and quality control, Golgi processing and vesicle traffic, Lysosomal sorting and endocytosis, Exocytosis and secretion, Nuclear and mitochondrial import

Study guide

Every protein starts on a cytosolic ribosome, and its sorting signals decide where it goes. Proteins with no signal stay in the cytosol. Proteins with an N-terminal hydrophobic signal sequence enter the secretory pathway. The signal recognition particle (SRP) binds the signal as it leaves the ribosome, pauses elongation, and docks on the SRP receptor of the rough ER. The ribosome is handed to the Sec61 translocon, translation resumes, and the chain threads into the lumen, where signal peptidase removes the signal. Other proteins are imported after translation: nuclear proteins use NLSs and importins, mitochondrial proteins use presequences and TOM/TIM complexes, and peroxisomal proteins often use a C-terminal SKL signal.

Membrane proteins get their topology during translocation. A cleaved signal sequence followed by a hydrophobic stop-transfer segment produces a single-pass protein with its N-terminus in the lumen and C-terminus in the cytosol. Internal signal-anchor sequences can insert in either orientation depending on nearby charges, and alternating start- and stop-transfer segments make multipass proteins. Because the ER lumen becomes the outside of the cell after vesicle fusion, the luminal side of a protein ends up facing outward at the plasma membrane.

In the ER lumen, chaperones such as BiP and the lectins calnexin and calreticulin help folding, and protein disulfide isomerase forms disulfide bonds. N-linked glycosylation begins when a 14-sugar oligosaccharide is transferred to asparagine. Proteins that cannot fold are sent back to the cytosol and destroyed by the proteasome (ER-associated degradation). If misfolded proteins pile up, sensors such as IRE1, PERK and ATF6 trigger the unfolded protein response, which boosts chaperones and slows translation and, if stress persists, leads to apoptosis.

Vesicles carry cargo between compartments. COPII vesicles move cargo from the ER to the Golgi, COPI vesicles return escaped ER proteins (those ending in KDEL) and move material backward through the Golgi, and clathrin vesicles bud from the trans-Golgi network and plasma membrane, pinched off by dynamin. Rab GTPases and tethers bring vesicles to the right target, and pairing of v-SNAREs with t-SNAREs drives fusion. The Golgi modifies glycans and adds O-linked sugars to serine and threonine as proteins pass from cis to trans. At the trans-Golgi network, proteins are sorted to lysosomes, regulated secretory granules, or the default constitutive secretion route.

Lysosomal hydrolases are tagged with mannose-6-phosphate in the cis-Golgi and bound by M6P receptors in the trans-Golgi network, which carry them to endosomes; acid pH releases them and the receptors recycle. Loss of M6P tagging (I-cell disease) causes hydrolases to be secreted. Material enters cells by endocytosis: phagocytosis of large particles, pinocytosis of fluid, and receptor-mediated endocytosis such as LDL uptake through clathrin-coated pits. Endocytosed cargo passes through early endosomes, where receptors are often recycled, to late endosomes and lysosomes. Exocytosis may be constitutive or regulated, with regulated release triggered by signals such as Ca²⁺.

Key ideas

• Sorting signals in a protein's sequence determine its destination.
• SRP targets ribosomes making signal-sequence proteins to the ER during translation.
• Start- and stop-transfer sequences set the topology of membrane proteins.
• The ER folds, glycosylates and quality-checks proteins, and stress triggers the unfolded protein response.
• COPII moves cargo forward, COPI retrieves, and clathrin works at the trans-Golgi and plasma membrane.
• M6P tags send hydrolases to lysosomes, and secretion is the default for unsignaled ER proteins.
• Nuclear and mitochondrial proteins are imported after translation using NLSs with Ran-GTP and presequences with TOM/TIM.

Formulas and rules

Common mistakes

• Assuming proteins need a signal to be secreted; secretion is the default for soluble ER proteins.
• Mixing up COPI and COPII directions.
• Thinking mitochondrial proteins use SRP; they are imported after translation.
• Forgetting that the ER luminal face becomes the extracellular face of plasma membrane proteins.
• Confusing N-linked glycosylation (asparagine, ER) with O-linked (serine/threonine, Golgi).

Before the test, make sure you can

• Explain how SRP and the translocon target and translocate a secretory protein.
• Predict membrane protein topology from its signal and stop-transfer segments.
• Describe ER folding, glycosylation, ERAD and the unfolded protein response.
• Match COPI, COPII and clathrin to their routes.
• Explain M6P sorting and predict the effects of I-cell disease.
• Describe receptor-mediated endocytosis of LDL and constitutive vs regulated exocytosis.
• Compare nuclear, mitochondrial and ER import mechanisms.
• Interpret pulse-chase, cell-free and mutant experiments on the secretory pathway.

Words to know

Signal sequence

A short stretch of hydrophobic amino acids, usually at the N-terminus, that targets a protein to the ER.

SRP

Signal recognition particle; binds signal sequences, pauses translation and brings the ribosome to the ER.

Sec61 translocon

The protein channel in the ER membrane through which polypeptides enter the lumen.

Stop-transfer sequence

A hydrophobic segment that halts translocation and becomes a transmembrane anchor.

BiP

An Hsp70-family chaperone in the ER lumen that helps proteins fold.

Unfolded protein response

A signaling program triggered by misfolded ER proteins that raises chaperones and slows translation.

ERAD

ER-associated degradation; misfolded proteins are returned to the cytosol and destroyed by the proteasome.

N-linked glycosylation

Addition of an oligosaccharide to asparagine in the ER lumen.

COPII

Coat protein complex that forms vesicles carrying cargo from the ER to the Golgi.

COPI

Coat protein complex for retrograde transport from the Golgi to the ER and between Golgi cisternae.

KDEL

A C-terminal sequence that causes soluble proteins to be retrieved to the ER.

Clathrin

A triskelion protein that forms cage-like coats on vesicles from the trans-Golgi and plasma membrane.

Dynamin

A GTPase that pinches off budding vesicles at their necks.

SNAREs

Paired membrane proteins (v- and t-SNAREs) that drive vesicle fusion with a target membrane.

Rab GTPase

A small G protein that marks membrane identity and recruits tethering factors.

Mannose-6-phosphate

A sugar tag added in the Golgi that targets hydrolases to lysosomes.

Receptor-mediated endocytosis

Uptake of specific ligands bound to receptors that cluster in clathrin-coated pits.

Nuclear localization signal

A short basic amino acid sequence that directs proteins into the nucleus via importins.

Sample practice questions

30 more questions like these, from level 1 to 5, are in the practice set.