Protein sorting
Protein Sorting & Vesicular Transport -
Complete Study Guide
🎯 THE BIG PICTURE
Proteins are made in the cytosol but need to get to different destinations. Think of it like a
postal system - each protein has an "address label" (signal sequence) that tells it where to go,
and different "delivery methods" get them there.
📦 THREE MAIN TRANSPORT MECHANISMS
1. Nuclear Pores (Nucleus)
● Selective gates - like airport security
● Actively transport large molecules
● Allow free diffusion of small molecules
2. Protein Translocators (ER, Mitochondria, Chloroplasts)
● Proteins cross membranes through translocator channels
● Like threading a needle - protein goes through a hole
3. Transport Vesicles (ER → Golgi → Plasma Membrane)
● Vesicles = tiny bubbles that bud off and fuse
● Ferry proteins between compartments
● Like delivery trucks carrying packages
🔵 NUCLEAR IMPORT (Post-translational)
Key Players:
● NLS = Nuclear Localization Signal (the "address")
● Importin = delivery truck protein
● Ran-GTP/GDP = energy switch
The Process (5 Steps):
1. Protein with NLS binds to importin in cytosol
2. Importin carries protein through nuclear pore
3. Inside nucleus, Ran-GTP binds importin → releases protein
4. Ran-GTP-importin exits nucleus
5. GTP hydrolysis (→GDP) releases importin for reuse
💡 Key Point: Proteins made on FREE ribosomes → transported AFTER synthesis
(post-translational)
🔴 MITOCHONDRIAL IMPORT (Post-translational)
Signal Sequence:
● Amphipathic N-terminal sequence (has both hydrophobic + hydrophilic sides)
● Like a molecular "key" - removed after entry by signal peptidase
Key Players:
● TOM = Translocase of Outer Membrane
● TIM = Translocase of Inner Membrane
● mtHsp70 = mitochondrial heat shock protein (chaperone)
● Signal peptidase = cuts off signal sequence
Energy Requirements (SUPER IMPORTANT):
1. Proton gradient (H⁺) across inner membrane = electrical potential
2. ATP for chaperone function
The Process:
1. Protein synthesized in cytosol as unfolded precursor
2. Signal sequence recognized by TOM receptors
3. Protein threaded through TOM complex (outer membrane)
4. At contact sites, passes through TIM complex (inner membrane)
5. Proton gradient pulls protein in (like a magnet)
6. mtHsp70 chaperone grabs protein using ATP, helps it fold
7. Signal peptidase clips off signal sequence
8. Protein folds into 3D structure in matrix
💡 Key Point: Proteins transported UNFOLDED, then folded by chaperones. Needs BOTH
membrane potential AND ATP!
🟡 PEROXISOMAL IMPORT
What are Peroxisomes?
● Organelles that do β-oxidation (break down fatty acids)
● Contain catalase enzyme (breaks down H₂O₂)
Unique Feature:
● Import proteins AND lipids from cytosol
● Proteins can be imported folded (unlike mitochondria)
● Signal sequence: PTS1 or PTS2 (peroxisomal targeting signal)
🟢 ENDOPLASMIC RETICULUM (ER) - Co-translational
Signal Sequence:
● Hydrophobic N-terminal (~20 amino acids)
● Recognized DURING translation
Key Players:
● SRP = Signal Recognition Particle (recognizes signal)
● SRP receptor on ER membrane
● Translocon = channel/plug in ER membrane
● Signal peptidase = cuts signal sequence
The Process (Co-translational = DURING synthesis):
1. Ribosome starts making protein
2. Signal sequence emerges from ribosome
3. SRP binds signal → pauses translation
4. SRP brings ribosome to SRP receptor on ER
5. Ribosome docks on translocon channel
6. Translation resumes, protein feeds through translocon into ER lumen
7. Signal peptidase cleaves signal sequence
8. Protein released into ER lumen or embedded in membrane
💡 Key Point: ER import happens WHILE protein is being made (co-translational). Ribosomes
attach to ER = Rough ER (RER)
🧬 MEMBRANE PROTEIN INSERTION
Type 1: Cleavable Signal (e.g., LDL Receptor)
● Signal sequence at N-terminus
● Signal cleaved off
● Protein ends up with N-terminus in ER lumen
Type 2: Internal Signal (Start-Transfer)
● Hydrophobic transmembrane domain acts as signal
● NOT cleaved
● Can have multiple passes through membrane (~7 helices for some proteins)
● Creates transmembrane proteins
Orientation Rules:
● Positive charges (Lys, Arg) stay on cytosolic side
● Hydrophobic segments span membrane
● Stop-transfer signals halt threading
🔧 ER PROTEIN QUALITY CONTROL
Protein Folding in ER:
● Chaperones help fold proteins correctly
● Glycosylation = adding sugar groups to asparagine (N-linked)
● Oligosaccharides added via dolichol carrier
Quality Sensors:
● Check for exposed hydrophobic regions (shouldn't be exposed if folded right)
● Check proper disulfide bonds
ERAD System:
● ERAD = ER-Associated Degradation
● Misfolded proteins → tagged with ubiquitin
● Sent to proteasome for destruction
● Keeps defective proteins from leaving ER
🟠 SMOOTH ER (SER)
Functions:
● Lipid synthesis (phospholipids, steroids, cholesterol)
● No ribosomes (smooth surface)
● Detoxification in liver
Phospholipid Synthesis:
● Made on cytosolic side of ER membrane
● Flippase enzyme flips lipids to other side
● Creates balanced bilayer
📬 VESICULAR TRANSPORT (ER → Golgi → Destination)
The Secretory Pathway:
ER → cis-Golgi → medial-Golgi → trans-Golgi → Vesicles → Destination
Golgi Functions:
● cis face (receiving end) - receives from ER
● medial (middle) - modifies proteins
● trans face (shipping end) - sorts and sends out
Modifications in Golgi:
● Glycosylation (add/modify sugars)
● Phosphorylation
● Proteolytic cleavage
● Sulfation
🎯 PROTEIN SORTING SIGNALS
Lysosome Targeting:
● Mannose-6-Phosphate (M6P) tag added in Golgi
● M6P receptors recognize tag
● Directs protein to lysosome (acidic pH ~4.5-5)
Default Pathway:
● No signal = goes to plasma membrane or secreted
Retention Signals:
● KDEL sequence = stay in ER
● Retrieved from Golgi back to ER if accidentally leave
🚛 VESICLE MOTOR PROTEINS
On Microtubules:
● Kinesin = moves vesicles OUTWARD (+ end, toward plasma membrane)
○ "Kick out"
● Dynein = moves vesicles INWARD (- end, toward nucleus)
○ "Pull back"
Vesicle Coating:
● COPII = ER → Golgi (anterograde)
● COPI = Golgi → ER (retrograde)
● Clathrin = Golgi → endosomes/lysosomes
🧪 EXPERIMENTAL APPROACH (How We Know This)
Palade's Pulse-Chase Experiment:
1. Give cells radioactive amino acids (pulse)
2. Track where labeled proteins go over time
3. Showed: ER → Golgi → secretory vesicles → outside cell
Cell Fractionation:
● Break cells, separate organelles by density
● Dense = RER (has ribosomes)
● Less dense = SER (no ribosomes)
● Proved different compartments exist
⚡ ENERGY REQUIREMENTS SUMMARY
Transport Energy Source When
Nuclear GTP (Ran-GTP) Post-translational
Mitochondrial ATP + Proton gradient Post-translational
ER GTP (SRP, translation) Co-translational
Vesicular ATP (motor proteins) Post-translational
🎓 QUICK REVIEW - MUST KNOW FOR 100%
Post-translational Transport:
● Nucleus: NLS + importin + Ran-GTP
● Mitochondria: Amphipathic signal + TOM/TIM + ATP + proton gradient + unfolded
● Peroxisomes: PTS signal + can be folded
Co-translational Transport:
● ER: Hydrophobic signal + SRP + translocon + signal peptidase
Key Differences:
● Nucleus & Mitochondria = proteins made FIRST, then transported
● ER = proteins transported WHILE being made
● Mitochondria = must be unfolded
● Peroxisomes = can be folded
Signal Sequences:
● NLS = basic amino acids (Lys, Arg rich)
● Mitochondrial = amphipathic α-helix, N-terminal
● ER = hydrophobic, N-terminal (~20 aa)
● Peroxisomal = SKL (Ser-Lys-Leu) at C-terminus
Vesicular Transport Order:
ER → cis-Golgi → medial-Golgi → trans-Golgi → vesicles → plasma
membrane/lysosomes/secretion
Quality Control:
● ER = ERAD (ubiquitin + proteasome)
● Golgi = adds M6P for lysosomal proteins
💯 SUMMARY OF EVERYTHING
We covered how cells sort proteins to the right location. Proteins start in the cytosol and need
"address tags" (signal sequences) to reach their destination.
Nuclear proteins use NLS signals and importin carriers, powered by Ran-GTP. Mitochondrial
proteins have amphipathic signals, get threaded through TOM/TIM complexes while unfolded,
and need both ATP and the proton gradient to enter. ER proteins have hydrophobic signals
recognized during synthesis by SRP, which brings ribosomes to the ER translocon channel.
The secretory pathway moves proteins from ER through the Golgi (where they're modified with
sugars and sorted) to final destinations via vesicles. Motor proteins (kinesin and dynein) move
vesicles along microtubules. Quality controlensures only properly folded proteins proceed -
misfolded ones are destroyed via ERAD.
Each pathway uses different signals, timing (co- vs post-translational), and energy
sources, but all ensure proteins reach their correct cellular address!
🔑 KEYWORDS TO MEMORIZE
● NLS - Nuclear Localization Signal
● TOM/TIM - Translocases (mitochondria)
● SRP - Signal Recognition Particle
● Translocon - ER protein channel
● Amphipathic - Has hydrophobic + hydrophilic regions
● Co-translational - During synthesis
● Post-translational - After synthesis
● ERAD - ER-Associated Degradation
● M6P - Mannose-6-Phosphate (lysosome tag)
● Chaperone - Protein folding helper
● Signal peptidase - Cuts signal sequences
You've got this! 🚀