BIOB10 FINALLL

Unfolded Protein Response (UPR)

• What it is: Emergency system in the ER when too many proteins are misfolded/unfolded.

• Causes: stress, mutations, or too much protein production.

• What happens:

  • Make more chaperones (helpers that fold proteins)

  • Make more proteases (cut up bad proteins)

  • Slow down new protein synthesis (so ER isn’t overloaded).

• Sensors: Proteins in ER that stay OFF until misfolded proteins show up.

  • Chaperones normally sit on these sensors (keeping them off).

  • When misfolded proteins appear → chaperones leave to help them → sensors turn ON → UPR begins.

CFTR (Cystic Fibrosis Protein)

• What it is: A chloride ion channel in epithelial cells.

• Mutation: In cystic fibrosis, the gene is faulty → CFTR misfolds.

• Misfolded CFTR gets tagged by ERAD (quality control system) → degraded → no chloride channel → symptoms of cystic fibrosis.

Coat Proteins & Vesicle Formation

• General role: Make membranes curve and form vesicles (cargo bubbles).

• Also grab cargo proteins for transport.

• The type of coat proteins present on a vesicle target it to specific organelles.

• Types:

  • COP II → ER → Golgi (forward/anterograde).

  • COP I → Golgi → ER (backward/retrograde).

  • Clathrin → sorting proteins to PM, lysosomes, endocytosis.

Clathrin Vesicles

• Made of triskelions (3 heavy + 3 light chains).

• They form a basket around vesicles.

• Need Adaptor Proteins (e.g. AP2) to connect clathrin shell to membrane.

  • Bind clathrin and form inner coat between clathrin shell and vesicle membrane.

PIPs (Phosphoinositidyl Phosphates)

• Special lipids that tell vesicles where to go.

• Different organelles have different PIPs → recruit different adaptors/coats.

• Example: AP2 binds PIPs + cargo receptors → activates clathrin → forms coated pit → pulls stuff into cell.

Vesicle Budding & Fission

• BAR proteins → bend membrane.

• Dynamin → protein that makes a "collar" around budding vesicle neck → cuts it off (fission).

• Shaping: Dimers bend membrane.

• Fission proteins: Cut vesicle loose.

• Dynamin (GTPase): Forms collar at vesicle neck, hydrolyzes GTP → pinches vesicle off.

Targeting → 3 Key Proteins:

1. Coat recruitment GTPases → start vesicle coating.

2. Rab proteins → guide vesicle to target.

3. SNAREs → dock & fuse membranes.

Coat-Recruitment GTPases (SAR1 → COPII)

1. SAR1 recruits COPII proteins to ER.

2. SAR1 switches GDP → GTP with GEF help → inserts into membrane → bends it.

3. Recruits other COPII proteins (SEC23, SEC24, SEC13, SEC31).

   • SEC23 and 24 bind ER transmembrane receptors on the cytosolic side to recruit cargo proteins. SEC13 and 31 join to form outer structural layer 

4. Vesicle forms → pinched off → coat removed after.

5. GTP hydrolysis → Sar1 + coat proteins released.

Golgi Apparatus

• Location: Near nucleus.

• Structure: Stacks of cisternae (pancakes).

• Compartments:

  • Cis-Golgi → sorting, phosphorylation, mannose removal.

  • Medial-Golgi → mannose removal, GlyNAC addition.

  • Trans-Golgi → sorting to final destination, adds Gal + NANA, sulfation.

Vesicle Targeting + fusion

1. Vesicle Tethering

• Tethering Factors: help vesicle get close to target membrane.

• First contact between vesicle + target membrane.

• Uses long fibrous proteins / tethering complexes from the target membrane.

2. Vesicle Docking

• v-SNARE (vesicle) + t-SNARE (target) = Vesicle is pulled in closer + aligned with target membrane.

3. Vesicle Fusion

• Actual merging of vesicle membrane with target membrane.

• Contents are released into the target compartment.

• SNARE proteins help push out water and bring membranes close enough to fuse.

• NSF protein unzips SNAREs afterwards so they can be reused..

Key players in vesicle targeting

Rab Proteins (small GTPases)

• Sit on vesicle membranes via lipid anchors.

• Each Rab = specific organelle identity.

• Recruit tethering factors to guide vesicle to the right place.

• Example: Rab5 → recruits more Rab5 (positive feedback = more recruitment).

SNARE Proteins

• v-SNARE = on vesicle.

• t-SNARE = on target membrane.

• Function: lock together → bring membranes close → fusion.

• After fusion: NSF separates SNAREs for recycling.

ER Retrieval

• ER → Golgi (Forward/Anterograde)

  • Vesicles from ER fuse together (after losing COPII coat) → form vesicular tubular clusters → fuse into ERGIC (ER-Golgi Intermediate Compartment).

    Golgi → ER (Backward/Retrograde)

  • Sometimes ER proteins accidentally leave → need retrieval.

  • Done by COPI-coated vesicles.

  • Signal sequences:

    • Membrane proteins: KKXX (Lys-Lys-X-X) on C-terminal.

    • Soluble proteins: KDEL (retrieved by KDEL receptor in Golgi).

Golgi Apparatus

• The cell’s processing & shipping center.

• Cis side → receives ER vesicles.

• Trans side → ships proteins out to final destination.

• Two models:

  • Vesicle transport: vesicles carry stuff forward.

  • Cisternal maturation: cisternae themselves move forward and mature.

Protein Modifications (Golgi Work)

1. Cleavage, sorting, modification.

2. Glycosylation

   • Protein modification that involves the addition of sugar molecules to proteins, lipids or other organic molecules.

Types:

• Complex N-Glycosylation (most common)

  • Sugars trimmed + new ones added

  • Steps: remove 3 Man → add GlcNAc → remove 2 Man → add more sugars.

• High Mannose Oligosaccharides

  • Only trimmed, no new sugars added.

• O-Linked Glycosylation

  • Sugars attached to Ser/Thr (not Asn).

  • Happens after folding, purpose unclear.

Polarized Structure of the Golgi

• Hypothesis 1: Vesicle Transport

  • Cisternae (Golgi stacks) are stable.

  • Vesicles carry proteins between cisternae.

  • Cisternae don’t move, but their composition changes.

• Hypothesis 2: Cisternal Maturation

  • New cisternae form from ER vesicles.

  • Cisternae move from cis → trans side.

  • Resident proteins get recycled backwards via COPI vesicles.

  • Evidence: Blocking ER→Golgi transport makes Golgi disappear.

Secretion of Proteins from the Golgi

Golgins = cytosolic tethering proteins.

• Long proteins in Golgi → tether vesicles (work w/ Rab).

• If phosphorylated → Golgi fragments/disperses. → ensures Golgi is distributed during mitosis

Lysosomes & Vacuoles

• Lysosomes: acidic, digestive organelles (enzymes = acid hydrolases).

• Proton pumps (V-type ATPases) keep inside acidic.

• Acidic pH (pH ~4.5) maintained by Vacuolar H⁺-ATPases (proton pump)

• Vacuoles (plants) = plant lysosomes.

Protein Sorting to Lysosomes

Lysosomal Enzymes

1. Synthesized + N-glycosylated in ER.

2. Sent to Golgi via COPII vesicles.

3. Cis-Golgi: mannose residues phosphorylated → M6P signal.

4. M6P recognized by Mannose-6-Phosphate Receptors (MPRs) in trans-Golgi.

5. Enzymes → clathrin vesicles → endosomes → lysosomes.

Vacuolar Enzymes (plants)

• Have Vacuolar Targeting Sequence (VTS).

• Recognized at trans-Golgi, sorted into vesicles.

• Like lysosomes in plant cells in that they also store hydrolytic enzymes. Have VTS.

Lysosomal Storage Disorders

• Cause: enzymes don’t reach lysosomes → buildup of undegraded stuff.

• I-cell disease: defective phosphorylation → no M6P signal → swollen lysosomes + junk builds up→ severe tissue + skeletal issues.

Cell Delivery & Transit

Exocytosis (stuff out)

• Vesicles fuse w/ PM → release contents outside cell.

• Glycosylation sites always face outside cell.

Constitutive Pathway

• Constitutive Secretion (Default Pathway)

• Involves the continuous unregulated synthesis and secretion of proteins.

Regulated Pathway

• Only release when triggered (ex. calcium in neurons).

• Proteins to be secreted are densely packaged into secretory granules that wait for a cellular signal before they release their contents.

• Involves proteins aggregating for entry into secretory vesicles

Synaptic Vesicles (Special Exocytosis)

• Docking: v-SNARE (Synaptobrevin) binds t-SNARE (Syntaxin).

• Priming I: SNAREs pull closer, SNAP25 binds.

• Priming II: Complexin locks SNARE complex → waiting.

• Trigger: Ca²⁺ binds Synaptotagmin.

• Fusion pore opening → release contents.

• SNAREs reset for reuse.

• Specialized class of small secretory vesicles. Synaptic vesicles dock at the plasma membrane and undergo a priming step to prepare for rapid fusion using SNARE-binding protein, v-SNARE and t-SNARE.

Endocytosis (stuff in)

• Bringing materials into the cell.

• Bidirectional trafficking decides fate of cargo.

Types of Endosomes

• Early endosome: sorting newly internalized stuff.

• Recycling endosome: sends proteins back to PM.

• Late endosome: fuses w/ lysosome.

• Multivesicular bodies (MVBs) → intermediate stage, formed via microtubules.

• Endolysosome: late endosome fused w/ lysosome.

Types

• Pinocytosis: cell "drinking," small continuous uptake.

• Caveolae: tiny cave-like invaginations.

• Macropinocytosis: big gulps, actin-driven protrusions.

• Phagocytosis: "cell eating," large objects, done by immune cells.

• Autophagy: cell eats its own old/damaged parts → fuses w/ lysosome

Receptor-Mediated Endocytosis (Example: LDL/Cholesterol)

• Involves cell surface receptors binding specific cargo for internalization.

• LDL (cholesterol carrier) binds LDL receptor → clathrin vesicle → endosome/lysosome → cholesterol released.

• Receptors recycled to membrane.

Cargo Receptor Fates

• Recycling to the plasma membrane for reuse.

• Degradation in lysosome,

• Transcytosis: receptors are recycled to a different plasma membrane, transporting cargo across the cell.

Pinocytosis ("cell drinking")

• Continuous formation of small endosomes.

• Mainly clathrin-coated vesicles or caveolae.

• No change in cell surface area.

Macropinocytosis

• Clathrin-independent.

• Defense mechanism (to clear toxins).

• Like pinocytosis but on a larger scale.

• Triggered by signalling pathways → actin makes membrane ruffles.

• Ruffles fold back + trap lots of fluid → form a macropinosome.

• Macropinosome fuses with endosome → cargo digested → cell reuses pieces.

Phagocytosis ("cell eating")

• Done by specialized cells (macrophages).

• Engulf bacteria/foreign bodies.

• Some pathogens escape by blocking fusion with lysosomes (e.g., TB bacteria).

Autophagy ("self-eating")

• Cell digests its own parts.

• Nonselective: random cytoplasm chunks.

• Selective: specific organelles/cargo.

• Uses autophagosomes → fuse with lysosomes.

• Steps:

  1. Bad organelle/protein wrapped in autophagosome.

  2. Autophagosome fuses with lysosome.

  3. Digested + recycled into building blocks.

Plasma Membrane & Transport

Main Function: barrier + selective exchange.

• Diffusion: high → low concentration, faster if small & nonpolar.

• Facilitated diffusion: via channel proteins.

• Transporters: bind specific molecules, change shape to move them.

Transport Types

1. Diffusion (passive)

   • High → low concentration.

   • Smaller + lipid-soluble (nonpolar) molecules cross membranes faster.

2. Transporters

   • Bind specific solutes strongly → Change shape → release solute to other side.

   • Can be passive (no energy) or active (needs energy).

   • Example: Glucose transporters regulated by insulin.

   • Low blood sugar = GLUT4 stuck in vesicles.

   • High blood sugar = insulin released → signals GLUT4 to move to PM → glucose uptake ↑

   • Type II diabetes = GLUT4/insulin signaling defects.

3. Channels

   • Weak interaction with solutes.

   • Form a pore (can open/close).

   • When open, solute diffuses through.

   • Can be gated:

     • Voltage-gated (Open/close based on electrical difference (membrane potential).

     • Ligand-gated (binding molecule).

     • Mechanically gated (stretch/pressure).

Insulin and GLUT4 Regulation

• When blood sugar level is low, insulin levels are also low and GLUT 4 remains in cytoplasmic vesicles

• Few GLUT transporters are on the on PM and there is very little internalization of glucose into cells.

• When blood sugar levels are high, insulin is secreted into the bloodstream

• Insulin binds to insulin receptors on PM which signals vesicles containing GLUT to exocytose GLUT4 into the PM and more transporter on PM means more internalization of glucose into cells.

Selectivity Filter

• Narrow part of a channel.

• Forces ions to lose water + line up in single file.

• Only specific ions/molecules fit → makes channels selective.

• Transport through channels = facilitated diffusion.

Active Transport

• Move ions uphill (low → high concentration).

• Require energy (ATP, light, redox, or coupled transport).

• Still bind molecules + change shape like carriers.

• P-type pumps: phosphorylate themselves during transport.

  • Example: Na⁺/K⁺ ATPase (3 Na⁺ out, 2 K⁺ in).

• ABC transporters: transport organic molecules (largest family).

• V-type pumps: proton pumps → acidify organelles.

• F-type pumps: opposite of V-type → use H+ gradient to make ATP using proton gradient.

Sodium-Potassium Pump (Na⁺/K⁺-ATPase)

How it works (step by step):

1. Pump grabs 3 Na⁺ (sodium) from inside the cell.

2. Pump gets phosphorylated (ATP donates a phosphate → energy!).

3. Pump changes shape → kicks out the 3 Na⁺ to the outside.

4. Pump then grabs 2 K⁺ (potassium) from outside the cell.

5. Pump gets dephosphorylated (phosphate leaves).

6. Pump resets back to its original shape → drops 2 K⁺ inside the cell.

---

Result:

• Always 3 Na⁺ out : 2 K⁺ in each cycle.

• Creates:

  • More Na⁺ outside the cell.

  • More K⁺ inside the cell.

• This sets up the membrane potential (electrical charge difference).

• It’s crucial for nerve impulses, muscle contraction, and cell volume control.

Transport in Long-Term Potentiation (Memory)

Ligand-gated ion channels:

• AMPA receptor: glutamate-gated → fast excitatory transmission.

• NMDA receptor: requires glutamate AND depolarization → Ca²⁺ influx → strengthens synapses (basis of learning/memory).

General Features Mitochondria

• Origin: Evolved from prokaryotes (endosymbiosis).

• DNA & Ribosomes: Have their own, but most genes moved to nucleus.

  • Only make ~1% of their proteins themselves.

  • The rest = made in cytosol, then imported post-translationally.

• Protein Import: Proteins don’t fold before transport. Cytosolic chaperones keep them unfolded until receptors pull them in.

• Internal membranes: Increase surface area for reactions.

Mito and Chloro

• Mitochondria: make ATP by oxidative phosphorylation (burning food molecules).

• Chloroplasts: make ATP by photosynthesis (using sunlight).

• Both:

  • Found in eukaryotes.

  • Likely came from bacteria (endosymbiont theory).

  • Have their own DNA + ribosomes (but most genes moved to nucleus).

  • Double membranes + special inner membranes full of protein machines for ATP.

  • Use electron transport chains (ETC) → pump protons → create electrochemical gradient → drive ATP synthase (chemiosmosis).

Chemiosmotic Coupling

• Definition: Using a proton (H⁺) gradient + charge difference across a membrane to power ATP synthesis.

• Basically: pump protons → store energy → let them flow back through ATP synthase → make ATP.

• Establishing an electrochemical gradient (generating differences in pH and/or membrane potential) that drives an energy-requiring process.

Functions - Mitochondria

• Energy production

  • Mitochondria: food → H₂O + CO₂ + ATP.

  • Chloroplasts: H₂O + CO₂ + light → food.

• How they make ATP:

  • Electron transport chain (ETC).

  • Chemiosmotic coupling = proton gradient drives ATP synthase.

Structure - Mitochondria

• Porins: protein channels in outer membrane → let small molecules in/out.

• Cristae: folds of inner membrane → increase surface area for ATP production.

• Binary fission: how mitochondria divide (like bacteria).

• OMM (Outer Membrane)

  • 50% lipids, 50% proteins.

  • Permeable to small molecules/ions (because of porins).

  • No electrochemical gradient here.

• IMM (Inner Membrane)

  • Folded into cristae (increase surface area).

  • 25% lipids, 75% proteins (lots of pumps/channels).

  • Impermeable (controls what enters/exits).

  • Contains pumps, channels, ATP synthase.

  • Special lipid: cardiolipin (stabilizes membrane like cholesterol does in PM).

  • Steep gradient.

• Spaces:

  • IMS (Intermembrane space): between OMM + IMM.

  • Matrix: inside IMM, contains DNA + ribosomes.

Dynamics (Fission & Fusion) - Mitochondria

• Fission (splitting)

  • The process by which mitochondria divide similar to binary fission

  • The mitochondria and ER are in close contact

  • ER tubules form a collar around the mitochondrion and initiate constriction which is completed by cytosolic proteins similar to dynamin.

  • This is a hydrolysis -driven constriction.

• Fusion (merging)

  • The process by which two mitochondria can fuse to form one larger mitochondrion.

  • OMM GTPases create a complex of proteins from which two OMMs can be fused.

  • Requires GTP and proton gradient

  • IMM fusion requires dynamin-related proteins and is also a GTP hydrolysis mediated process

• Fission: one mitochondrion splits into two. ER helps squeeze → dynamin-like proteins cut it. GTP hydrolysis powers final cut.

• Fusion: two mitochondria merge. Needs GTP + proton gradient. Outer membranes fuse first (requires GTP + proton gradient). Inner membranes fuse second (needs dynamin-like proteins).

Stop-transfer sequence

A protein signal sequence that halts translocation through a membrane channel.

Protein Targeting & Import

Proteins for mitochondria are made in cytosol → imported using signal sequences.

Signal Sequences

• Matrix proteins: have +charged N-terminal sequence.

• IMM proteins: have internal targeting + stop-transfer sequences.

  1. OMM Proteins

• TOM: main entry gate.

• SAM: folds & inserts proteins into OMM.

• MIM1: alternative import for some OMM proteins, imports proteins that skip TOM.

2. IMM Proteins

• TIM23: moves proteins into IMM or matrix.

• TIM22: for transporters & energy-related proteins, imports IMM proteins with internal signals

• OXA: inserts proteins either from TIM23 or those made inside mitochondria.

Example: Matrix Protein Transport

1. Signal sequence recognized by TOM.

2. Goes through TOM → chaperones fall off.

3. Moves into TIM23 → pulled into matrix.

4. Signal sequence cleaved → protein folds properly.

• Import receptors on the outer mitochondrial membrane (OMM) bind the signal sequence on proteins.

• Protein enters through the TOM complex (goes signal sequence first).

• Chaperones (helper proteins) detach.

• Protein threads through the TIM23 complex in the inner mitochondrial membrane (IMM) into the matrix.

• The positively charged signal sequence helps drive movement into the matrix.

• Signal peptidase cuts off the signal sequence.

• The protein then folds into its mature form (can even start folding while still entering).

Insertion routes - IMM

🔹 IMM Insertion Routes

• Route 1: TOM → TIM23 → stop-transfer → stuck in IMM.

• Route 1 (alt): TOM → TIM23 → OXA inserts into IMM.

• Route 2: TOM → TIM22 → inserts transport proteins into IMM.

• TOM → TIM23 pathway.

  • Signal sequence guides the protein inside.

  • Signal sequence is cleaved in the matrix.

  • A stop-transfer sequence halts the movement.

  • TIM23 moves the protein sideways (laterally) into the IMM.

• OXA-dependent insertion

  • Protein goes TOM → TIM23 → matrix.

  • Stop-transfer sequence is recognized by OXA.

  • OXA moves the protein laterally into the IMM.

IMM Protein Transport – Route 2

• Protein has an internal signal sequence instead of an N-terminal one.

• Goes TOM → TIM22.

• TIM22 recognizes the internal signal and laterally inserts the protein into the IMM.

Insertion routes - OMM

🔹 OMM Insertion

• TOM → protein held by chaperones → SAM inserts + folds into OMM.

🔹 Proteins from mtDNA

• Made in matrix → use OXA to insert into IMM.

• Some OMM proteins can’t stop-transfer through TOM.

• They pass fully into the intermembrane space first.

• Then interact with SAM complex, which inserts and folds them into the OMM.

mtDNA Protein Transport

• Proteins made inside the mitochondria (from mtDNA + ribosomes in the matrix).

• Still need OXA to insert them into the IMM.

Protein Domain

• A domain is a distinct, stable part of a protein.

• Folds independently.

• Usually has a specific function (like a binding site or catalytic region).

Cellular Respiration - Glycolysis

Glycolysis (cytosol)

• Breaks glucose → 2 pyruvate.

• 10 step reaction

• Net: +2 ATP, +2 NADH, +2 pyruvate.

• Anaerobic: fermentation (lactate in muscles / ethanol in yeast).

• Aerobic: pyruvate → Acetyl-CoA → enters mitochondria.

Cellular Respiration - Link RXN

Link Reaction

• Pyruvate enters mitochondria → converted to Acetyl-CoA.

• Fatty acids can also → Acetyl-CoA (via beta-oxidation).

Cellular Respiration - Citric Acid Cycle (Krebs Cycle, TCA)

• Acetyl-CoA + oxaloacetate → citrate → back to oxaloacetate.

• Net per turn: +3 NADH, +1 FADH₂, +1 ATP.

Cellular Respiration - Electron Transport Chain (ETC)

• NADH + FADH₂ donate high-energy electrons to chain of proteins.

• Complexes I, III, IV pump protons → create H⁺ gradient.

• O₂ = final electron acceptor → makes H₂O.

• Proton-motive force used by ATP synthase.

ATP Synthase

Parts of ATP Synthase

1. F₀ complex – sits in the inner membrane (IM), acts like a proton channel. Protons flow through it from the intermembrane space → matrix.

2. F₁ complex – sticks into the matrix, looks like an orange sliced into segments. This is where ATP is actually made.

How ATP is Made

• Protons move down their gradient through F₀.

• This makes F₀ and F₁ rotate relative to each other.

• Rotation changes the shape of F₁ and drives ADP + phosphate → ATP.

Cristae & Efficiency

• ATP synthase forms dimers at the cristae ridges.

• Proton pumps are right nearby, so protons quickly reach ATP synthase.

• This arrangement makes ATP production more efficient.

• F₀ part: proton channel in IMM.

• F₁ part: catalytic head in matrix.

• Protons flow through F₀ → rotation → F₁ makes ATP from ADP + Pi.

• Cristae ridges concentrate proton flow → ↑ efficiency.

DNP (Deadly Diet Drug)

• Uncouples ETC + ATP synthase.

• Protons leak back across IMM → ATP not made.

• ETC keeps burning fuel to restore gradient → hypermetabolic state.

• Symptoms: rapid weight loss, fatigue, overheating, death.

Structure - Chloroplast

• Big: size of a human red blood cell.

• 3 membranes + 3 spaces (like mitochondria but w/ extra thylakoids):

  • Grana: stacks of thylakoids.

  • Stroma lamellae: bridge-like thylakoids connecting grana.

  • Outer envelope membrane: permeable, has porins.

  • Inner envelope membrane: impermeable, packed with transporters.

  • Thylakoids: flattened sacs in stroma, where light reactions happen. 75% protein, very dense/gel-like.

• Spaces inside:

  • Intermembrane space (between outer + inner membranes).

  • Stroma:

    • Has DNA + ribosomes.

    • Many enzymes for carb synthesis.

  • Thylakoid space (inside thylakoid).

Fission + Fusion - Chloroplast

• Fission: uses FtsZ (like bacterial division). Makes a contractile ring.

• Fusion: two chloroplasts merge like mitochondria.

Protein Targeting & Import (Chloroplasts)

1. Transport Gateways

• TOC = outer membrane gate

• TIC = inner membrane gate

• Proteins pass through these to get inside the chloroplast.

• TOC (outer) + TIC (inner): transport proteins into stroma.

  2. Stroma Proteins (inside the main chloroplast space)

  • Protein has a cTP “address tag” → recognized by TOC.

  • Then interacts with TIC → pulled into stroma.

  • Transit peptide cut off by a protease.

  • Chaperones fold the protein into its working shape.

  • Tells protein "go to stroma."

  3. Thylakoid Lumen Proteins (inside thylakoid sacs)

  • Proteins first go to stroma.

  • Then special thylakoid transfer signals guide them into the thylakoid lumen.

  4. Thylakoid Membrane Proteins

  • Can use Sec pathway, OXA-like pathway, or TAT pathway to get inserted into the thylakoid membrane.

Protein Translocation (importing proteins into chloroplasts)

• Most proteins are made in the cytosol (free ribosomes).

• Signal sequence = Chloroplast Transit Peptide (CTP).

🔹 TOC Complex = Translocon of Outer Membrane of Chloroplast.
🔹 TIC Complex = Translocon of Inner Membrane of Chloroplast.

• Steps:

  1. Protein + transit peptide brought by chaperones.

  2. CTP binds to TOC receptor.

  3. Stroma-targeting domain interacts w/ TIC → ATP/GTP used for import.

  4. Protease cuts off CTP.

  5. Protein is refolded by chaperones.

  6. If a thylakoid transfer domain is exposed → protein goes to thylakoid.

Pathways for thylakoid targeting

1. Sec pathway (like Sec system in bacteria):

   • Needs ATP + H+ gradient.

   • Produces soluble proteins.

2. OXA-like pathway:

   • Needs GTP + H+ gradient.

   • Uses alternative thylakoid signal.

   • Produces membrane proteins (inserted into thylakoid).

3. TAT (Twin-Arginine Translocation):

   • Signal has 2 arginines.

   • Needs H+ gradient.

   • Produces soluble proteins.

Photosynthesis

Equation:
6CO₂ + 6H₂O (+ light) → C₆H₁₂O₆ + 6O₂

Light Reactions ("photo part")

• Location: thylakoid membrane.

• Function: Photosynthetic electron transfer.

• Steps:

  • Water split by reaction center → releases O₂, protons, and electrons.

  • Photon excites electron in chlorophyll → electron goes through ETC.

  • ETC pumps protons → makes H+ gradient.

  • Gradient drives ATP synthase → makes ATP.

  • Final products = ATP + NADPH + O₂ - splitting two H2O molecules

Dark Reactions ("synthesis part")

• Location: stroma.

• Function: Carbon fixation (Calvin Cycle).

• Steps:

  • Use ATP + NADPH from light reactions.

  • Converts CO₂ → glyceraldehyde-3-phosphate (G3P), a 3-carbon sugar.

  • Steps:

    1. Carboxylation: CO₂ joins RuBP → unstable 6C compound → splits into G3P. (Enzyme: RuBisCO)

    2. Reduction: G3P is energized using ATP/NADPH.

    3. Regeneration: RuBP is rebuilt to keep the cycle going.

  • Summary: 3 CO₂ + 6 NADPH + ATP → 1 G3P

  • G3P exported → cytosol → makes glucose, sucrose, other organics.

Collaboration with Mitochondria

• G3P leaves chloroplast → cytosol → used to make glucose, sucrose, starch, fats.

• These molecules can later be broken down in mitochondria via glycolysis & TCA cycle to release energy (ATP).

• So: Chloroplasts capture energy, mitochondria release it for work.

Cytoskeleton

Functions

• Structure: scaffolding, organizes organelles.

• Shape: maintains cell shape.

• Functions:

  • Keeps the cell’s shape.

  • Helps the cell move.

  • Moves organelles and stuff inside the cell.

Types

1. Actin filaments (microfilaments).

2. Microtubules.

3. Intermediate filaments.

• Built by noncovalent bonds → allows rapid assembly/disassembly.

Nucleation

• First step in building a filament.

• A small “seed” cluster of molecules forms → other molecules stick to it and grow a bigger structure.

• Initial step in forming a new structure where a small, stable cluster of molecules (a "nucleus") forms

• Nucleation is the formation of the initial oligomer which can be used for elongation.

Oligomer

• A tiny chain made of a few monomers (small building blocks).

• Forms right after nucleation.

Actin Filaments

• Actin = a protein that makes filaments.

Structure

• Filament = double helix w/ twist every 37 nm.

• Polarity:

  • Barbed end (+) = fast growing.

  • Pointed end (−) = slow growing.

• Diameter = 8 nm.

• If many actin monomers around → growth at both ends.

• If few monomers → growth mostly at plus end.

• Each actin monomer starts with ATP, but after joining the filament, ATP → ADP.

Forms

• G-actin = monomer (globular).

• F-actin = polymer (filamentous).

Actin Filament Depolymerization

• Breaking down actin filaments by removing monomers (mostly at the minus end).

• Actin filaments are always being built and broken down → very dynamic.

Actin Filament Treadmilling

• Monomers are added at plus end and removed at minus end.

• Makes it look like the filament is “moving” in one direction, even though it’s just recycling.

Actin-Binding Proteins

🔥 Nucleating Proteins

• Kickstart actin filament growth.

• Examples: Arp2/3 complex, Formin.

🧲 Monomer Binding Proteins

• Grab actin monomers and either block growth or help growth.

🚦 Capping Proteins

• Bind to plus end or minus end of F-actin to prevent growth or shrinkage and regulate length of filaments.

• Control filament length.

• “Critical concentration”: the rate of subunit growth is equal to rate of subunit loss (no net filament growth) = balance point → filament stays same size.

• Uncapped filaments polymerize and depolymerize more rapidly than capped filaments.

🧱 Side-Binding Proteins

• Stick to the side of filaments.

• Stabilize and stiffen the actin filament and prevent other interactions.

✂ Severing Proteins

• Cut filaments into smaller pieces.

• Example: Cofilin (also twists actin to weaken it).

🔗 Cross-Linking Proteins

• Connect filaments into stable networks.

• Add support to the cell.

📎 Plasma Membrane Binding Proteins

• Connect actin filaments to the plasma membrane.

• Important for cell movement.

ARP 2/3 Complex

• Works like a starter kit for actin filaments.

• Mimics the plus (“barbed”) end of actin to help new filaments grow from scratch.

• Stays attached to the minus end, letting the filament grow quickly at the plus end.

• Can attach to the side of existing filaments to create branches at a 70° angle.

• Needs NPFs (Nucleation Promoting Factors) to work.

• Catalyzes nucleation by grabbing G-actin.

Formins

• Dimers that wrap around + end → stabilize & promote growth.

• Make long, unbranched actin filaments and bundle them in parallel.

• Usually anchored at specific spots on the plasma membrane → focused filament growth.

• Stay attached to the plus end, so filaments keep elongating without losing subunits.

• Overall: makes straight, stable filaments grow fast.

Profilin

• Binds G-actin monomers at - end.

• Blocks - end binding, leaves + end free → promotes growth at + end.

• Helps elongate actin filaments.

• Binds actin monomers at the minus end → directs addition to plus end.

• Prevents random polymerization at the minus end.

• After a monomer is added, profilin detaches and grabs the next monomer, speeding up growth.

Thymosin

• Similar to profilin, but instead of promoting growth, it sequesters monomers → prevents/slows filament growth.

Side-Binding Proteins

• Example: Tropomyosin → stabilizes filaments & regulates other protein binding.

Cofilin

• A filament severing protein.

• Twists actin filaments to create mechanical stress → makes them weaker and easier to break.

• Helps recycle actin monomers and remodel the cytoskeleton.

  • Prefers binding to old F-actin (ADP-actin).

• Gelsolin → severs AND caps ends to prevent re-growth.

Cross-Linking Proteins

• Fimbrin → tight packing.

• Alpha-actinin → loose packing.

• Filamin → 3D web structures.

Myosin (Motor Protein)

• Motor proteins that “walk” along actin using ATP.

• Usually move cargo or cause contraction.

🧱 Myosin Structure

• Made of 2 heavy chains (tail + heads) and 4 light chains.

• Heavy chains form bipolar filaments:

  • Heads face outward.

  • Middle part (bare zone) has no heads.

• Can be inactive (heads folded back) until activated.

• Movement: always towards the + end of actin.

Conventional Myosin (Myosin II)

• Forms bipolar filaments (tails in middle, heads on outside).

• Has bare zone in the middle with no heads.

• Resting state: heads bent, inactive.

🔄 Myosin Motor Activity

• Myosin uses ATP in a cycle:

  1. ATP binds → myosin releases actin.

  2. Hydrolysis → myosin head “cocks.”

  3. Reattachment → phosphate leaves.

  4. Power stroke → pulls actin.

  5. ADP leaves → rigor state (until new ATP binds).

• Bipolar arrangement → pulls actin filaments from both sides → contraction.

Functions:

• Non-muscle cells: motility, cytokinesis, stress fibers.

• Muscle cells: contraction.

Bipolar Filaments (Myosin II)

• Filaments with motor heads at both ends pointing opposite ways.

• Can pull actin filaments toward the center from both sides, generating contraction.

• Important for cell movement, shape changes, and tension generation.

Muscles & Sarcomere Structure

• Muscle fiber = long multinucleated cell (fusion of myofibrils).

• Myofibrils = repeating sarcomeres.

Sarcomere Anatomy

• Repeating units of actin + myosin filaments in muscle cells.

• Z disk → ends of sarcomere; actin + ends attach here.

• M line → center; anchors myosin.

• I band → actin only.

• A band → overlap region (actin + myosin).

• H zone → myosin only (middle of A band).

• Relaxed: wide I-band + H-zone.

• Contracted: sarcomere shorter, I & H shrink, filaments slide (length doesn’t change).

Actin Filament Regulators

• CapZ: caps the plus (+) end of actin → stops it from shrinking.

• Tropomodulin: caps the minus (-) end → also prevents shrinking.

• Alpha-actinin: cross-links actin filaments → keeps them organized.

• Tropomyosin & Nebulin: bind along the sides of actin → stabilize filaments.

Myosin & Sarcomere Proteins

• Titin: helps position thick filaments and acts like a spring during contraction/relaxation.

• T-tubules: membrane folds that wrap around myofibrils, helping signals reach deep into the muscle fiber.

• Tropomyosin: covers actin’s binding site → blocks myosin from grabbing actin when muscle is relaxed.

• Troponin: attached to tropomyosin; when Ca²⁺ binds, it moves tropomyosin away → myosin can bind actin → muscle contracts.

Unconventional Myosin (Myosin V)

• A special type of myosin with 2 heads and a long neck → can take long steps along actin without letting go.

• Has more light chains (6 pairs).

• Main job: moves vesicles and organelles inside the cell.

• Helps transfer cargo to synapses and makes sure organelles are divided correctly when cells split.

• Function: intracellular transport.

  • Heads bind actin.

  • Tails bind cargo (vesicles, organelles).

  • Moves towards + end using ATP.

Cell Cortex & Protrusions

• Cell cortex: dense actin network just under the membrane → supports the cell and helps it move.

Types of protrusions:

• Pseudopodia: thick, 3D projections → help the cell move.

• Filopodia: thin, spike-like → sense environment and guide movement.

• Lamellipodia: flat, sheet-like → push the cell forward.

• Blebs: bubble-like protrusions → form when the membrane detaches slightly from actin to push forward.

Steps in Cell Movement

1. Protrusion: actin polymerization at + end

2. Attachment: protrusion sticks to surface.

3. Pulling: bulk of cell moves forward (driven by myosin II).

Cell Migration

• Mesenchymal Migration:

  1. Cell pushes lamellipodia forward.

  2. Lamellipodia sticks to surface.

  3. Back of cell contracts forward.

• Lamellipodia Formation:

  • WASP proteins → activate Arp2/3 → new branched actin → pushes membrane forward.

• Regulation (Rho GTPases):

  • Cdc42 → sets cell polarity.

  • Rac and WAVE → promotes lamellipodia (via Arp2/3 to nuclear branched actin filaments).

  • Rho → activates formin (inactivates cofilin) + myosin II → stress fibers + contraction.

Front vs Back Pathways

• Front (Rac-GTP):

  • Activates WAVE → Arp2/3 → actin branching.

  • Blocks myosin activity (no contraction here).

• Back (Rho-GTP):

  • Activates formin → unbranched actin.

  • Activates myosin II → contraction + stress fibers.

Bacteria Hijacking Actin

• Listeria monocytogenes:
  Uses ActA protein to activate Arp2/3 → grows actin tails → rockets around cell.

• Salmonella:
  Injects proteins → activates WASP/Arp2/3 → lamellipodia engulf it → infection.

• Enteropathogenic E. coli:
  Injects proteins → activates Arp2/3 → makes actin pedestals under itself → destroys microvilli → bad digestion.

Diseases

• Duchenne Muscular Dystrophy (DMD):
  Missing dystrophin, so actin not anchored to membrane → fragile muscle cells → muscle weakness.

• Griscelli Syndrome:
  Missing Myosin Va → melanosomes (pigment carriers) stuck in cells → albinism.

Microtubule Terms

• Polymerization: adding monomers → filament grows.

• Depolymerization: removing monomers → filament shrinks.

• Catastrophe: sudden switch from microtubule growth → shrinkage.

• Rescue: switch from microtubule shrinkage → growth.

• Perinuclear: “around the nucleus.”

Centrioles & Centrosome

• Centriole:

  • Tiny barrel-shaped structure made of microtubules.

  • Always in pairs → together they form the centrosome.

  • The centrosome is the cell’s "microtubule organizer," kinda like the manager telling microtubules where to grow.

• Pericentriolar material:

  • "Around the centriole."

  • Cloud of proteins around centrioles.

  • Acts like an anchor point for microtubules to start growing.

Microtubules (MTs)

• Protofilament: a chain of tubulin pairs (α-tubulin at the “-” end, β-tubulin at the “+” end).

• Microtubule: made of 13 protofilaments lined up side by side → forms a hollow tube with polarity.

• Not a perfect cylinder → slight stagger.

• Uniform polarity (all + ends same side).

Growth & Shrinkage (Dynamic Instability)

• Microtubules grow by adding GTP-tubulin at the + end → forms a stable GTP cap.

• After incorporation, GTP → GDP → less stable → if the cap is lost, microtubule shrinks quickly.

• This grow-shrink cycle allows microtubules to remodel fast.

Depolymerization Hypothesis

• GTP-β-tubulin: keeps microtubules straight and stable.

• GDP-β-tubulin: makes microtubules curve and unstable, prone to breaking down.

Nucleation

• Needs high subunit concentration.

• To start building a microtubule, cells use:

  • γ-TuRC (γ-tubulin ring complex) → acts like a "base cap" for the (-) end.

  • (+) end grows outward.

• In animal cells: happens at the centrosome (MTOC = microtubule-organizing center).

• Plants & fungi have other MTOCs too.

Microtubule Accessory Proteins

• Nucleation: γ-TuRC, augmin, stathmin.

• Capping: +TIPs.

• Stabilization: XMAP215.

• Severing: katanin.

• Bundling: MAP2, Tau, Plectin.

Stabilizing Proteins (Tau, MAP2)

MAPs (Microtubule-Associated Proteins)

• MAPs = proteins that bind microtubules to stabilize them.

• 2 domains: MT binding + filament binding.

• Tau = keeps them tightly packed.

• MAP2 = allows looser spacing.

Nucleating Proteins (MAPs, Augmin):

• MAPs = general regulators.

• Augmin = helps γ-TuRC attach to existing microtubules → creates branching MTs.

+TIPs (Plus-End Tracking Proteins)

• Stick to growing (+) end.

• Some cause catastrophe (shrinkage, e.g., Kinesin-13).

  • Catastrophe Factors

    • Promote MT depolymerization at + end.

    • Example: Kinesin-13 (bends protofilaments apart).

• Some promote growth (e.g., XMAP215 delivers tubulin).

  • XMAP215

    • Opposite of catastrophe factors.

    • Adds tubulin to + end → promotes growth.

Sequestering Protein (Stathmin)

• Grabs free tubulin and "hides" it → prevents polymerization.

• When phosphorylated (activity inhibited) → stops hiding tubulin.

Severing Protein (Katanin)

• Cuts microtubules by breaking protofilament bonds.

• Katanin (“katana”) = cuts all 13 protofilaments.

• Can cause catastrophe OR repair (if β-tubulin replaces hole, growth continues).

Motor Proteins

• Use ATP to “walk” along MTs.

• Dynamic MTs: used for motility.

• Stable MTs: used for organelle positioning.

• Microtubule motor proteins transport cargo:
  Kinesins move toward the (+) end (anterograde).
  Dyneins move toward the (-) end (retrograde).

Dynein

• Direction: Move toward (-) end (inward, retrograde).

• Functions: spindle positioning, organelle transport, vesicle/endosome movement.

• Structure:

  • 2 heavy chains, intermediate + light chains.

  • ATP-binding head.

  • MT-binding stalk.

• Movement:

  1. Head binds MT, tail binds cargo.

  2. ATP binds → MT domain releases.

  3. Linker-swing” motion: ATP binding swings head forward, hydrolysis + release = power stroke (~8 nm step).

  4. ATP hydrolysis → power stroke moves cargo 8 nm.

Kinesin

• Motor proteins that usually move toward the + end of microtubules (some exceptions like Kinesin-14 go toward - end).

• Structure (tetramer, 4 parts):

  • Head: binds microtubules + uses ATP to generate movement.

  • Neck: directs the “walking” direction.

  • Stalk: flexible connector.

  • Tail: binds cargo (vesicles, organelles).

• Movement:

  1. Rear head (ATP-bound) = strongly attached.

  2. Front head (ADP-bound) = loosely attached.

  3. ATP hydrolysis in rear → swings forward.

  4. Neck linker shifts → heads swap roles.

• Movement: “walking” along microtubules using ATP-driven steps.

Adaptor Proteins

• Connect motors to their cargo.

• Dynein adaptor: dynactin stabilizes dynein-cargo, links multiple dyneins for stronger transport.

• Kinesin adaptors: attach cargo like mitochondria, peroxisomes, secretory vesicles.