Source: Cell Biology, University of Florida | Chapter 15
Tags: organelles, protein sorting, signal sequence, endoplasmic reticulum, ER, Golgi apparatus, nuclear pore, vesicular transport, secretory pathway, endocytosis, exocytosis, SNARE, clathrin, proteasome, lysosome, SRP, BIOL 101
Difficulty: Intermediate to Advanced | Prerequisites: Chapter 7 (translation, ribosomes), Chapter 11 (membrane structure, lipid bilayer)
A eukaryotic cell is divided into membrane-enclosed compartments, each with a specialised function. This chapter explains how proteins, which are almost all made on ribosomes in the cytosol, get to the right compartment. The answer involves signal sequences (postal codes built into the protein's amino acid sequence) and three transport mechanisms: gated transport through nuclear pores, transmembrane transport via protein translocators (for ER, mitochondria, peroxisomes), and vesicular transport (for moving cargo between the ER, Golgi, plasma membrane, lysosomes, and endosomes). Understanding this chapter ties together transcription, translation, membrane structure, and cell signalling. You should be comfortable with ribosomes, translation, and lipid bilayer basics before starting.
Proteins are sorted to the correct organelle by signal sequences in their amino acid sequence. They enter the nucleus through nuclear pores (folded), cross into the ER during translation (co-translational import via SRP), or unfold to cross into mitochondria. Once inside the ER, proteins travel onward by vesicular transport: small membrane-bound vesicles bud from one compartment and fuse with the next, delivering cargo through the secretory pathway (ER to Golgi to plasma membrane) or the endocytic pathway (plasma membrane to endosomes to lysosomes).
Signal sequence (sorting signal)
A stretch of amino acids (typically 15 to 60 residues) within a protein that specifies its destination organelle. Different signal sequences direct proteins to different compartments. Often removed once the protein has been sorted. Think of it as: a postal code embedded in the protein itself that tells the cell where to deliver it.
Nuclear pore complex
A large structure that perforates the nuclear envelope, allowing bidirectional transport between nucleus and cytosol. Small molecules pass freely; large molecules (proteins, RNA, ribosomal subunits) require a sorting signal and active transport.
Nuclear localisation signal (NLS)
A signal sequence (typically rich in positively charged lysines and arginines, located internally in the protein) that directs a protein from the cytosol into the nucleus via nuclear pores. The protein remains folded during transport.
Nucleoporins
The approximately 30 different proteins that make up the nuclear pore complex. Disordered segments form a tangled meshwork inside the pore that blocks large molecules but allows small, water-soluble molecules to pass. They also contain binding sites for nuclear import receptors.
Nuclear import receptor
A soluble cytosolic protein that binds both the nuclear localisation signal on a cargo protein and the nucleoporins of the pore complex, guiding the cargo through the pore. Dissociates from cargo once inside the nucleus and returns to the cytosol.
Signal recognition particle (SRP)
A cytosolic particle (part RNA, part protein) that binds to both the ribosome and the ER signal sequence as it emerges from the ribosome. SRP slows translation until the ribosome docks at the ER membrane via the SRP receptor.
SRP receptor
A protein embedded in the ER membrane that recognises SRP, accepts the ribosome, and passes it to a protein translocator in the ER membrane. SRP is then released and recycled.
ER signal sequence
A segment of 8 or more hydrophobic amino acids, usually at the N-terminus, that directs a protein to the endoplasmic reticulum for co-translational import. Cleaved by signal peptidase after translocation for soluble ER proteins.
Protein translocator
A channel in the ER (or mitochondrial) membrane through which a polypeptide chain is threaded during or after translation. For the ER, the translocator opens when the ER signal sequence binds and closes after translocation is complete.
Stop-transfer sequence
A second hydrophobic sequence within a polypeptide that halts translocation through the ER translocator, causing the protein to be released sideways into the lipid bilayer. The result is a transmembrane protein anchored in the ER membrane.
Start-transfer sequence (internal signal sequence)
An internal hydrophobic sequence that initiates translocation of a polypeptide loop into the ER lumen without being cleaved. Works in combination with stop-transfer sequences to produce multipass transmembrane proteins.
Glycosylation (N-linked)
The covalent attachment of a preformed branched oligosaccharide (14 sugars) to asparagine residues (at Asn-X-Ser/Thr sequences) on proteins entering the ER lumen. Catalysed by oligosaccharyl transferase. Functions: protects proteins from degradation, retains them in ER until properly folded, serves as a transport signal.
Dolichol
A specialised lipid in the ER membrane to which the preformed oligosaccharide is attached before being transferred to a protein during N-linked glycosylation.
Chaperone proteins (ER)
Proteins in the ER lumen (e.g. BiP) that bind to misfolded or partially assembled proteins, preventing them from leaving the ER until they are properly folded. Properly folded proteins are released and transported onward in vesicles.
Unfolded protein response (UPR)
A cellular stress response triggered when misfolded proteins accumulate excessively in the ER. The UPR slows synthesis of new proteins and stimulates the cell to expand its ER and increase production of chaperones.
KDEL sequence
A four-amino-acid C-terminal retrieval signal (Lys-Asp-Glu-Leu) on soluble ER-resident proteins. If these proteins escape to the Golgi, the KDEL sequence is recognised by a receptor that packages them into COPI-coated vesicles for retrograde transport back to the ER.
KKXX sequence
A C-terminal retrieval signal (two lysines plus any two amino acids) on ER-resident transmembrane proteins. Binds directly to COPI coat proteins for retrograde transport back to the ER.
Golgi apparatus
A stack of flattened membrane cisternae with two distinct faces: the cis face (receiving side, adjacent to ER) and the trans face (shipping side, toward plasma membrane). Proteins pass through the Golgi via vesicles and are further modified (sugars added, removed, or trimmed) and sorted to their final destinations.
Transport vesicle
A small membrane-bound sphere that buds from one compartment and fuses with another, delivering both soluble cargo and membrane components. Proteins remain folded during vesicular transport.
Clathrin
A coat protein that forms basket-like cages on the cytosolic surface of budding vesicles at the Golgi and plasma membrane. Clathrin itself does not select cargo; that job belongs to adaptins.
Adaptin
A protein that links the clathrin coat to the vesicle membrane and selects cargo molecules by binding to cargo receptors. Different adaptins operate at different membranes (Golgi vs plasma membrane), reflecting different cargo.
Dynamin
A GTP-binding protein that assembles as a ring around the neck of a clathrin-coated pit and constricts it, pinching the vesicle off from the parent membrane.
COPII-coated vesicle
A vesicle coated with COPII proteins that buds from the ER and travels to the Golgi (anterograde transport).
COPI-coated vesicle
A vesicle coated with COPI proteins that buds from the Golgi and returns to the ER (retrograde transport), retrieving escaped ER-resident proteins.
Rab proteins
Small GTPases on the surface of transport vesicles that serve as molecular identity markers. Each organelle and vesicle type carries a unique combination of Rab proteins. Recognised by tethering proteins on the target membrane.
Tethering proteins
Proteins on the cytosolic surface of target membranes that grab incoming vesicles by binding to their Rab proteins, initiating the docking process.
SNAREs (v-SNARE and t-SNARE)
Transmembrane proteins on vesicles (v-SNAREs) and target membranes (t-SNAREs) that interact to dock the vesicle and catalyse membrane fusion, delivering the vesicle's cargo.
Constitutive secretion
The continuous, unregulated release of proteins from the cell by exocytosis. Operates in all cells.
Regulated secretion
The stimulus-dependent release of proteins stored in secretory vesicles. Occurs only in specialised secretory cells (e.g. hormone-secreting cells, neurons). Requires an extracellular signal to trigger exocytosis.
Endocytosis
The uptake of material from outside the cell by invagination of the plasma membrane to form vesicles. Includes pinocytosis (small vesicles, less than 150 nm, "cell drinking") and phagocytosis (large particles, greater than 250 nm, "cell eating," carried out by specialised phagocytic cells).
Receptor-mediated endocytosis
A specific form of endocytosis in which cell-surface receptors bind extracellular molecules (ligands), cluster in clathrin-coated pits, and are internalised. Example: LDL receptors bind LDL cholesterol, which is internalised in clathrin-coated vesicles and delivered to lysosomes for degradation.
Endosome
A membrane-bound sorting compartment that receives material from endocytic vesicles. In its acidic interior, ligands dissociate from receptors. Receptors are typically recycled to the plasma membrane; cargo is delivered to lysosomes for degradation.
Lysosome
A membrane-bound organelle containing hydrolytic enzymes (nucleases, proteases, glycosidases, lipases, phosphatases) that are active at acidic pH (approximately 5.0). The principal site of intracellular digestion. Receives material from endocytosis, phagocytosis, and autophagy.
Cytosol: enclosed by plasma membrane; site of most metabolic pathways, protein synthesis, and cytoskeletal assembly
Nucleus: surrounded by a double membrane (nuclear envelope); contains the genome, site of DNA and RNA synthesis; communicates with cytosol via nuclear pores
Endoplasmic reticulum (ER): the largest membrane system in the cell
Rough ER: studded with ribosomes, synthesises proteins destined for secretion, membranes, or other organelles
Smooth ER: lacks ribosomes, synthesises lipids and steroid hormones, metabolises carbohydrates, detoxifies drugs, stores calcium
Golgi apparatus: stacks of flattened cisternae; receives proteins and lipids from ER, modifies (adds/trims sugars), sorts, and dispatches them
Lysosomes: sacs of digestive enzymes for intracellular degradation
Endosomes: sorting stations for endocytosed material
Mitochondria: ATP synthesis by oxidative phosphorylation; double membrane
Peroxisomes: oxidative breakdown of toxic molecules, producing hydrogen peroxide
The nuclear envelope and ER likely evolved through invagination of the plasma membrane of an ancestral cell
Mitochondria (and chloroplasts in plants) originated by endosymbiosis: an ancestral eukaryotic cell engulfed an aerobic prokaryote
Virtually all proteins are synthesised by ribosomes in the cytosol. Their amino acid sequence contains sorting signals that direct them to the correct destination. Proteins lacking any sorting signal remain in the cytosol.
1. Gated transport through nuclear pores
Nuclear pores perforate the double membrane of the nuclear envelope
Small, water-soluble molecules diffuse freely through the pore
Large molecules (proteins destined for the nucleus, RNA and ribosomal subunits leaving the nucleus) require specific sorting signals and active transport
Proteins enter the nucleus folded, guided by nuclear import receptors that bind both the nuclear localisation signal (NLS, rich in lysines/arginines) and the nucleoporin fibrils extending from the pore
Inside the nucleus, import receptors release their cargo and return to the cytosol
Nuclear export works similarly: nuclear export signals (leucine-rich, internal) are recognised by export receptors that guide cargo out through the pore
The more transcriptionally active the nucleus, the more pores it has
2. Transmembrane transport via protein translocators
Used for import into the ER, mitochondria, chloroplasts, and peroxisomes
Proteins must be unfolded to thread through the translocator channel (except for ER import, which is co-translational)
Proteins are refolded after import
Import into the ER (co-translational):
An ER signal sequence (typically N-terminal, 8+ hydrophobic amino acids) emerges from the ribosome
SRP binds the signal sequence and the ribosome, pausing translation
SRP docks with its receptor on the ER membrane
The ribosome is handed off to a protein translocator; SRP is released and recycled
Translation resumes, and the polypeptide is threaded through the translocator into the ER lumen as it is synthesised
For soluble proteins: the N-terminal signal sequence is cleaved by signal peptidase; the completed protein is released into the ER lumen
For transmembrane proteins: a stop-transfer sequence (hydrophobic) halts translocation and anchors the protein in the bilayer. Internal start-transfer and stop-transfer sequences create multipass transmembrane proteins
The ER is the entry point for the entire endomembrane system: proteins destined for the Golgi, endosomes, lysosomes, and the cell surface all enter the ER first. Once inside, they never re-enter the cytosol
Import into mitochondria:
Mitochondria have a double membrane but cannot have pores (they need to maintain their proton gradient)
Mitochondrial signal sequences (N-terminal, rich in positively charged amino acids) are recognised by import receptors
The protein must be unfolded to pass through translocators in the outer membrane (TOM complex) and inner membrane (TIM complex)
The signal peptide is cleaved, and the protein refolds inside the matrix
3. Vesicular transport
Used for moving proteins between the ER, Golgi, endosomes, lysosomes, and plasma membrane
Transport vesicles bud from one compartment and fuse with the next
Proteins remain folded during transit
Both soluble cargo and membrane lipids/proteins are delivered
Vesicle budding is driven by assembly of a protein coat on the cytosolic surface
Clathrin-coated vesicles: bud from the Golgi (with adaptin 1, destination: lysosomes via endosomes) and from the plasma membrane (with adaptin 2, destination: endosomes)
COPII-coated vesicles: bud from the ER, travel to the Golgi (anterograde)
COPI-coated vesicles: bud from the Golgi, return to the ER (retrograde, retrieval pathway)
Clathrin forms the cage structure but does not choose cargo. Adaptins provide specificity: they link clathrin to the membrane and capture specific cargo molecules by binding cargo receptors
Dynamin (a GTPase) assembles around the neck of the budding vesicle and pinches it off
After budding, the coat is shed so the vesicle membrane can interact with its target
Each vesicle carries a unique combination of Rab proteins (GTPases) on its surface
Tethering proteins on the target membrane recognise and grab the vesicle via its Rab proteins
Once tethered, v-SNAREs on the vesicle interact with complementary t-SNAREs on the target membrane
SNARE pairing docks the vesicle in place and catalyses fusion of the two membranes, releasing the cargo
This multi-step recognition system ensures vesicles fuse only with the correct target
Chaperone proteins in the ER lumen bind misfolded proteins and prevent them from leaving
Only properly folded proteins are packaged into COPII vesicles for transport to the Golgi
Persistently misfolded proteins are translocated back to the cytosol and degraded by proteasomes
Example: in cystic fibrosis, a mutation in the CFTR chloride channel causes slight misfolding. The protein would function at the plasma membrane, but ER quality control retains and degrades it. The disease results from loss of functional protein at the cell surface, not from loss of protein activity per se
If misfolded proteins accumulate excessively, the unfolded protein response (UPR) is triggered: slows new protein synthesis, expands the ER, and boosts chaperone production
ER-resident soluble proteins carry a C-terminal KDEL sequence. If they escape to the Golgi, KDEL receptors capture them and package them into COPI vesicles for retrograde return to the ER
ER-resident transmembrane proteins carry a C-terminal KKXX sequence that binds directly to COPI coat proteins for retrograde transport
Disulphide bonds: formed in the ER lumen (not the cytosol, which is too reducing) by protein disulphide isomerase (PDI)
N-linked glycosylation: a preformed 14-sugar oligosaccharide, attached to dolichol in the ER membrane, is transferred en bloc to Asn-X-Ser/Thr sequences on newly translocated proteins by oligosaccharyl transferase. Functions: protects from degradation, retains in ER until folded, acts as transport signal
Oligosaccharide processing begins in the ER and continues in the Golgi, where sugars are added, removed, and trimmed as proteins pass through cisternae from cis to trans face
Proteins exiting the trans Golgi network are sorted: to lysosomes (via endosomes), to the cell surface (constitutive or regulated secretion), or returned to the ER (retrieval pathway)
Constitutive (default): continuous, unregulated exocytosis; delivers newly synthesised proteins and lipids to the plasma membrane in all cells
Regulated: proteins are stored in secretory vesicles until an extracellular signal triggers their release (e.g. hormone secretion, neurotransmitter release)
Phagocytosis ("cell eating"): large particles (greater than 250 nm) engulfed by specialised phagocytic cells (e.g. macrophages)
Pinocytosis ("cell drinking"): small vesicles (less than 150 nm) take up fluid and dissolved molecules
Receptor-mediated endocytosis: cell-surface receptors bind specific ligands, cluster in clathrin-coated pits, and are internalised
Example: LDL binds LDL receptors, internalised in clathrin-coated vesicles, vesicles lose coat and fuse with endosomes, in the acidic endosome environment LDL dissociates from receptor, LDL is delivered to lysosomes for degradation (releasing free cholesterol), LDL receptors are recycled back to the plasma membrane
Endosomes serve as a sorting centre for endocytosed material (analogous to the Golgi's role for the secretory pathway)
Receptor fates after endocytosis: recycled to the same membrane, degraded in lysosomes, or moved to a different membrane surface (transcytosis)
Contain a wide array of hydrolytic enzymes (active at pH 5.0, maintained by H+ pumps)
Receive material from endocytosis, phagocytosis, and autophagy (degradation of the cell's own damaged organelles)
Breakdown products (amino acids, sugars, nucleotides) are transported back to the cytosol for reuse
Students often think proteins travel to the ER after being fully made in the cytosol. For ER-destined proteins, import is co-translational: the ribosome docks at the ER membrane and the protein is threaded through the translocator as it is being synthesised.
Students often confuse the three transport mechanisms. Nuclear import uses pores (protein stays folded). ER/mitochondrial import uses translocators (protein must be unfolded for mitochondria, co-translational for ER). Vesicular transport uses membrane-bound vesicles (protein stays folded).
Students often think clathrin selects cargo. Clathrin forms the structural cage. Adaptins provide the cargo selectivity by binding cargo receptors.
Students often assume cystic fibrosis is caused by a non-functional CFTR protein. The mutant CFTR protein can function, but ER quality control recognises it as slightly misfolded and degrades it before it reaches the plasma membrane.
⚠️ Be able to describe the three mechanisms of protein sorting (gated nuclear transport, transmembrane transport, vesicular transport) and give examples of each.
⚠️ Know the sequence of events in co-translational ER import: signal sequence emerges, SRP binds, SRP docks with receptor, ribosome handed to translocator, translation resumes, signal cleaved.
⚠️ Understand how stop-transfer and start-transfer sequences produce single-pass and multipass transmembrane proteins.
⚠️ Know the coat proteins and their routes: COPII (ER to Golgi), COPI (Golgi to ER), clathrin (Golgi to lysosomes, plasma membrane to endosomes).
⚠️ Be able to explain how Rab proteins, tethers, and SNAREs ensure vesicles fuse with the correct target.
⚠️ Understand the cystic fibrosis example as a case where ER quality control is detrimental.
⚠️ Know the difference between constitutive and regulated secretion.
⚠️ Be able to trace the path of LDL through receptor-mediated endocytosis.
True or False: Proteins entering the nucleus must be unfolded to pass through nuclear pores.
Fill in the blank: The ________ binds to the ER signal sequence and the ribosome in the cytosol, pausing translation until the ribosome docks at the ER membrane.
True or False: COPII-coated vesicles travel from the Golgi back to the ER.
Fill in the blank: v-SNAREs on vesicles interact with ________ on the target membrane to dock and fuse.
True or False: In cystic fibrosis, the mutant CFTR protein is completely non-functional.
Answers: 1. False (nuclear import proteins remain folded; it is mitochondrial import that requires unfolding). 2. Signal recognition particle (SRP). 3. False (COPII goes ER to Golgi; COPI goes Golgi to ER). 4. t-SNAREs. 5. False (the protein can function, but ER quality control degrades it before it reaches the cell surface).
Q: What are signal sequences, and why are they important for protein sorting?
A: Signal sequences are stretches of amino acids (typically 15 to 60 residues) within a protein's primary structure that specify its destination organelle. Different signal sequences direct proteins to different compartments (nucleus, ER, mitochondria, peroxisomes). Without these signals, proteins remain in the cytosol. The signal is often removed after the protein reaches its destination.
Q: Describe the steps of co-translational protein import into the ER.
A: (1) The ribosome begins translating the mRNA and the N-terminal ER signal sequence emerges. (2) SRP binds the signal sequence and the ribosome, pausing translation. (3) SRP docks with the SRP receptor on the ER membrane. (4) The ribosome is transferred to a protein translocator; SRP is released. (5) Translation resumes, and the polypeptide is threaded through the translocator into the ER lumen. (6) The signal sequence is cleaved by signal peptidase (for soluble proteins), and the protein is released into the ER lumen.
Q: How do clathrin, adaptins, and dynamin work together in vesicle budding?
A: Cargo molecules bind to cargo receptors in the membrane. Adaptins bind the cytoplasmic tails of these receptors and recruit clathrin, which assembles into a basket-like cage on the cytosolic surface, shaping the membrane into a coated pit. Dynamin (a GTPase) forms a ring around the neck of the pit and constricts it, pinching the vesicle off from the parent membrane. After budding, the clathrin coat is shed.
Q: How does the cell ensure that transport vesicles fuse only with the correct target membrane?
A: Each vesicle carries a unique combination of Rab proteins (GTPases) on its surface. Tethering proteins on the target membrane recognise these Rabs and capture the vesicle. Once tethered, v-SNAREs on the vesicle interact with complementary t-SNAREs on the target membrane, docking the vesicle and catalysing membrane fusion. This multi-layered recognition system (Rab-tether, then SNARE-SNARE) provides specificity.
Q: Why is the cystic fibrosis CFTR example significant in the context of ER quality control?
A: The most common CF mutation (F508del) causes only a slight misfolding of the CFTR protein. The protein would function as a chloride channel if it reached the plasma membrane. However, ER quality control recognises the misfolded shape, retains it via chaperones, and ultimately sends it to the cytosol for proteasomal degradation. The disease occurs because functional protein is discarded before it can reach its destination, not because the mutation destroys the protein's activity. This illustrates how quality control, while normally beneficial, can sometimes be detrimental.
This chapter depends heavily on Chapter 7 (ribosomes, translation) and Chapter 11 (membrane structure, lipid bilayers, transmembrane proteins).
Vesicular transport and the secretory/endocytic pathways connect to cell signalling: receptors are delivered to the cell surface by exocytosis and removed by endocytosis, regulating the cell's sensitivity to signals.
The ER quality control and UPR are relevant to disease biology (cystic fibrosis, neurodegenerative diseases involving protein misfolding) and pharmacology (drugs that modulate ER stress pathways).
intracellular compartments, organelles, protein sorting, signal sequence, sorting signal, nuclear pore, nuclear localisation signal, NLS, nucleoporin, nuclear import receptor, endoplasmic reticulum, ER, rough ER, smooth ER, SRP, signal recognition particle, SRP receptor, protein translocator, co-translational import, ER signal sequence, stop-transfer sequence, start-transfer sequence, transmembrane protein, glycosylation, N-linked glycosylation, dolichol, oligosaccharyl transferase, chaperone, BiP, unfolded protein response, UPR, KDEL, KKXX, COPI, COPII, clathrin, adaptin, dynamin, Golgi apparatus, cis face, trans face, cisternae, transport vesicle, Rab protein, tethering protein, SNARE, v-SNARE, t-SNARE, vesicle docking, membrane fusion, constitutive secretion, regulated secretion, exocytosis, endocytosis, pinocytosis, phagocytosis, receptor-mediated endocytosis, LDL, endosome, lysosome, autophagy, cystic fibrosis, CFTR, mitochondrial import, TOM, TIM, protein folding, disulphide bond, PDI, BIOL 101, cell biology