Unit 2

Plasma Membrane

• Restricts passage of polar molecules

• Cells need to:

  • Ingest essential nutrients

  • Excrete metabolic waste

  • Regulate ion concentrations

• Use membrane proteins for cell import/export

Ion Gradients in Mammalian Cells

• Na+: Lower inside, higher outside

• K+: Higher inside, lower outside

• Ca2+: Lower inside, higher outside

Permeability of Membranes

• Hydrophobic molecules: O2, CO2, N2, Steroid hormones

  • Easily pass through

• Small uncharged polar molecules: H2O, urea, glycerol, NH3

  • Some leakage

• Large uncharged polar molecules: Glucose, sucrose

  • Very sparingly

• Ions: Na+, K+, H+, Ca2+

  • NO

Active vs. Passive Transport

• Active: Against concentration gradient, energetically unfavorable

  • Requires energy

• Passive transport: With concentration gradient, favorable

  • Can be with channel or transporter

  • Channel- Faster transport, weaker interaction with solute

• Both have high specificity

Electrochemical Gradients and Membrane Potential

• Electrochemical gradient has membrane potential, more favorable when they work together

  • When they oppose each other, high unfavorable

Transporters (carrier proteins)

• Expose bound solute to inside/outside

• Intermediate state= occluded (both sides closed)

• Vmax- Rate the carrier can flip between states

• Affinity for solute= Km

• Uniport- One transported molecule

• Symport- Transported molecule with transporter ion

• Antiport- Transported molecule moves in the opposite direction of transporter ion

Driving Active Transport

• Coupled transporter (secondary active transport)

  • Energy is used from favorable movement (moves in opposite directions, one favorable, one not)

• ATP-Driven Pump (Primary active transport)

  • ATP is hydrolyzed, phosphate drives molecule against electrochemical gradient

• Photosynthetic light driven pump

  • Energy from light drives molecule against electrochemical gradient

Distribution Gradients and Transcellular Transport

• Lumen

  • Low glucose concentration

  • Glucose and Na+ enter symport to epithelium

• Epithelium

  • High glucose concentration

  • Glucose passively transports to extracellular fluid (uniport)

  • Na+ enters Na+/K+ pump (antiport)

  • Na+ exits cell, K+ enters cell

• TIght junctions

  • Prevent solute flow

  • Act as transporter fences

ATPase Driven Pumps

• P-type pump

  • Ions (H+, K+, Na+, Ca2+)

  • ATP hydrolyzes, phosphate group drives ion across gradient

• ABC Transporter

  • Hydrolyzes 2 ATP, doesn’t use the phosphate

  • Transports small molecules

• V-type Proton Pump

  • Hydrolyzes ATP, H+ enters pump and crosses membrane

• F-Type ATP synthase

  • H+ enters pump

  • ADP + P are condensed to ATP from moving protein

  • Used in mitochondria

P-Type Ca2+ ATPase Pump

• In muscle cell sarcoplasmic reticulum (VERY unfavorable)

• Lumen side: Calcium binding cavity

• Cytosolic side: Nucleotide binding domain, ATP, Phosphorylation domain, phosphorylated aspartic acid, actuator domain

• E1: High Ca2+ affinity, 2 bound Ca2+

• E2: Low affinity for Ca2+, no bound Ca2+

• Pump cycle:

  • Open to cytosol side, 2H+ out, 2Ca2+ bind, ATP with no phosphate

  • Pump closes

  • ATP hydrolyzes, ADP is replaced with new ATP, phosphate group attached

  • Pump opens to lumen, 2Ca2+ leaves, 2H+ enters

  • Pump occludes with 2H+

  • Phosphate leaves

Na+/K+ Pump

• 3 Na+ out and 2 K+ in, against chemical gradients

• ATP hydrolyzes, phosphate group binds

• Na+ binding is favored in cytoplasm

ABC Transporters

• Bacterial

  • Cytosolic side: 2 ATPase domains

  • Extracellular: Closed solute binding site, hydrophobic domains

  • 2 ATP binds ATPase domains, solute enters domain

  • ATP hydrolyzes, solute exits into cytosol

• Eukaryotic

  • Solute is bound, ATPase domains are empty

  • ATP binds, solute exits cell

  • ATP hydrolyzes, transporter closes

• Examples:

  • MDR, TAP, CFTR

Channels

• Form pores across membranes

• Highly selective pores that open and close rapidly

• Most are ion channels, move quickly

• Always passive, down gradient

Aquaporins

• Move water in/out of cells

• Lines with polar AAs

• Allow passage of molecules, not ions

  • Hydrogen relay due to asparagine

• Moves ions with water

Ion-Channels

• Voltage gated

  • Open/close due to membrane potential

• Ligand gated

  • Ligand binding opens channel

  • Intra/extra cellular

• Mechanically gated

  • Signal opens/closes

K+ Channel Structure

• Selectivity filter, spaced carbonyls don’t allow Na+ to bind

• Selectivity loop is specified for K+ ions only

Energetics of Membrane Transport for Uncharged Molecules

• Molecules want to move down concentration gradient

• ΔGtransport depends on concentrations inside/outside cell

• ΔGtransport=RTln [S] inside/[S] outside

• ΔGtransport=ΔGchemical -ΔGmembrane

• ΔGtransport=RTln [S] inside/[S] outside +zFΔEm

• Em= electric potential difference between two compartments

Rough ER

Makes surface/non integral proteins

Smooth ER

Makes lipids, stores calcium

Golgi Apparatus

Packages/modifies proteins

Carbohydrate synthesis, sorting and dispatch station

Mitochondria

Make ATP

Lysosomes

Recycles proteins

Labyrinthine ER

Compromises 50% of membranes

Families of Organelles

1. Nucleus and cytosol

2. Secretory and endocytic

3. Mitochondria

4. Plastids (plants)

Topological Equivalency

• Space shares a continuous boundary between organelles

• If the pH’s differed, the protein would not be stable

• Lumens resemble extracellular environment

Gated Transport and Transmembrane

Gated transport is selective

Transmembranes use translocator proteins, topologically distinct

Signal Sequences

• Import into nucleus

  • NLS

  • ID by short + AA region

• Import into mitochondria

  • Alternate + and hydrophobic AA

• Import into peroxisomes

  • C term

  • SKL AAs

• Import into ER

  • N term

  • 5-10 hydrophobic AA

• Return to ER

  • C term

  • KDEL

Nuclear Import/Export

• Bidirectional

• Import- Histones, polymerases, transcription regulators, protein that work in nucleus

  • NLS signal binds

• Export- mature mRNAs destined for translation, made in nucleus, don’t work there

  • NES signal binds

Nucleoporins

• Unstructured: Tangle, kelp-like domain prevents passive diffusion of large molecules (because they can’t bind)

• Scaffold: Aid in bending membrane

• Nuclear Pore Complex (NPC)

  • Basket faces nuclear side

  • Selective

Ran GTPase

• Ran-GDP in cytosol

• Goes into nucleus, Ran-GEF switches GDP for GTP

• Ran-GTP exits nucleus into cytosol, Ran-GAP hydrolyzes GTP into GDP and releases Pi

Nuclear Import Workflow

• Can transport fully folded proteins (large pore)

• Importin is in the cytosol, high aff. for NLS, binds cargo

• Importin enters nucleus, Ran-GTP binds to importin and importin releases cargo into nucleus

  • Binding Ran-GTP reduces aff. for cargo

• Importin exits into cytosol and Ran-GDP unbinds to importin

Nuclear Export Workflow

• Exportin enter nucleus (not binded to anything)

• Ran-GTP binds to exportin, cargo then binds to exportin

  • Binding Ran-GTP increases aff. for cargo binding

• Exportin enters cytosol, Ran-GAP hydrolyzes Ran-GTP and exportin releases both Ran-GDP and cargo into cytosol

• Exportin is “all or nothing”, both Ran-GTP and cargo

Mitochondria vs Chloroplast

Mitochondria: Cross 1 or 2 membranes to get to matrix, outer or inner membrane

Chloroplast: Cross 1, 2, or 3 membranes to get to thylakoid space

Mitochondrial Protein Translocators

• TOM complex: From outer to intermembrane, only alpha helical

• SAM complex: Outer to intermembrane, b-barrels

• TIM22/23: Intermembrane to inner matrix

• OXA complex: Insert proteins made in mitochondria

Import into the Mitochondrial Matrix

• Precursor protein in cytosol (chaperones keep unfolded)

• Signal sequence of protein binds to TOM complex, TOM inserts protein into intermembrane space, TIM 23 pulls it through to matrix

• Protein properly folds in matrix, signal is cleaved by peptidase

Transport to Intermembrane Space

• Unfolded protein goes through TOM in outer membrane, goes partially through inner membrane with TIM 23

• Once signal sequence is through TIM23 and reaches stop sequence, signal sequence is cleaved

• Protein is properly folded and inserted on inner membrane with stop sequence bound

• Protein can remain bound or protease can cleave the stop sequence

Alternative Pathway for Inner Membrane Proteins

• Unfolded protein goes through TOM and TIM23, 1st signal sequence is cleaved

• Protein is in matrix, 2nd sequence binds to OXA complex (can also be a protein made in the matrix)

• OXA inserts protein on inner membrane

Chloroplast Import

• Precursor protein has chloroplast signal sequence and thylakoid signal sequence

• Protein binds to receptor in TOC complex

• Protein moves through TOC to TIC complex and enters stroma

• Chloroplast signal sequence is cleaved

• Thylakoid signal sequence takes protein into thylakoid, protein fold and releases sequence

Peroxisomes

• Contain oxidative enzymes

• Detoxifies harmful molecules

• Breakdown of fatty acids into Acetyl CoA

• Antioxidant- Absorbs reactive O2 species

Import into Peroxisomes

• SKL= peroxisome-targeting signal

• Import folded proteins

• SKL binds to cytosolic receptor, PEX5

• Peroxins form oligomeric pores, space in between ring of proteins

• Protein enters peroxisome, Pex5 receptor unbinds and leaves peroxisome via Pex 2,10,12 complex

Endoplasmic Reticulum

• Synthesis of membrane and secretory proteins

• Synthesis of lipids, in SER

• Ca+ storage

Import into ER

• Co-translational

  • ER bound ribosome is synthesizing protein and protein is being imported into ER

  • Why? The folded protein would not be stable in the cytoplasm

• Post-translational (less common)

  • Ribosome has finished creating protein and protein signal binds to receptor

SRP and ER Import Workflow

• Signal created from growing polypeptide, N-term

• Signal binds to SRP, pauses translation

• SRP takes peptide to bind to protein translocator, SRP binds to SRP receptor

• Once peptide is bounds to protein translocator, translation and translocation resume

• SRP is released from SRP receptor and is recycled

Free and Membrane Bound Ribosomes

• There is a common pool of ribosome units

• Ribosomes can stay in cytosol creating proteins

• OR they can bind to the ER lumen

• They all return to the common pool

Insertion of Single Pass Protein into ER Lumen

• Incomplete protein has both a start-transfer sequence and stop-transfer sequence

• Start transfer sequence is bound to translocator

• Protein is pulled through until the stop transfer signal is reached

• Signal peptidase moves the protein away from the translocator and onto the membrane

• Start transfer sequence is removed and stop transfer sequence stays attached

• Protein finished sequence on cytosolic side

Insertion of Single Pass Protein with Internal ER Signal Sequence

• N-term in cytosol, C-term in lumen

  • Start transfer sequence binds to translocator

  • Positively charged stays on cytosolic side, N term stays in cytosol and C-term is threaded through

• C-term in cytosol, N-term in lumen

  • Start transfer sequence binds to translocator

  • Positively charged stays on cytosolic side, C-term stays in cytosol and N-term is threaded through

Integration of Multi-Pass Proteins into ER

• Alternating start/stop transfer sequences that thread the protein through the membrane

• The first start transfer sequence determines the orientation of the entire protein

• Hydrophobic peaks indicate passes through the membrane

Insertion of Tail Anchored Proteins

• Recognition

  • Pre-targeting complex binds to start sequence and takes protein and binds it to Get3 ATPase

• Targeting

  • ATPase takes protein to oligomeric pore

  • Protein’s tail is then embedded onto ER lumen membrane facing the cytosol

• Release

  • ATPase hydrolyzes and separates from oligomeric pore

• Recycling

  • ATPase gets rid of ATP and Pi and gets a new ATP

ER Resident vs ER Transient Proteins

• ER Resident=return to ER

  • PDI, helps proteins form disulfide bonds, oxidize and isomerase mode

  • Ero1, re-oxidizes PDI

• ER resident proteins have KDEL sequence at C-term

• ER resident function= protein folding

• ER transient=Born in ER, don’t stay

  • No KDEL

Unfolded Protein Response

• Unfolded protein response activated by accumulation of unfolded proteins

• If not corrected, they will be degraded by proteasome in cytosol

• Sensors for misfolded proteins

  • IRE1, PERK, ATF6

• PERK

  • Phosphorylation inactivates translation initiation factor

  • Proteins reduced entering the ER

  • Selective translation of TRP2

• ATF6

  • Proteolysis in golgi releases and translated TRP3

IRE1

• Kinase domain with 2 phosphates and 2 kinases

• Ribonuclease domain splices introns out of pre-mRNA and fuses them

• mRNA is translated into transcription regulator protein

• Transcription regulator enters nucleus via importin and activates transcription of chaperone mRNA

• Chaperone mRNA enters cytosol and binds to SRP on ER lumen and is matured

• Chaperone helps correctly fold protein

Secretory Pathway

• Cells need to communicate, respond, and eat

• They use membrane systems to perform exo/endocytosis

• Lumens resemble the cell exterior

• Vesicle move proteins in containers

• Anterograde: Forward

• Retrograde: Backwards

Vesicular Transport

• Donor compartment begins budding a vesicle with contents selected for transport

• Transport vesicle is free in cytoplasm

• Vesicle fuses to target compartment and empties contents

Coated Vesicles Used for Trafficking

• Clathrin- From inside plasma membrane to early endosome, variable movement

• COPI- From golgi to ER, retrograde

• COPII- From ER to Golgi, anterograde

Clathrin Vesicles

• Triskelion formation allows mesh structure around vesicle

• Binds to AP2 on vesicles

• Workflow:

  • Assembly

    • Cargo receptor binds cargo on donor membrane, adaptor protein attaches to cargo receptor

  • Bud formation

    • Clathrin binds to adaptor protein

  • Vesicle formation

    • Clathrin triskelion forms, membrane bending occurs

    • Vesicle detaches from membrane

  • Once vesicle is free, clathrin detaches from vesicle

Dynamin

• Helps pinch off vesicles (clathrin-coated)

• Dynamin helix wraps around site of membrane bending

• GTPase protein that hydrolyzes and drives conformational change

Membrane Phosphoinositides

• Each membrane has its own PIPs

• Molecular “zip codes”

• Different PIPs: PI(3,5)P2

  • Phosphorylated in carbon position 3,5

  • P2 is 2 phosphates

• Different organelles have specific PIPs

  • PI(4,5)P₂ → plasma membrane

  • PI(3)P → early endosomes

  • PI(3,5)P₂ → late endosomes/lysosomes

  • PI(4)P → Golgi

  • PI(3,4,5)P₃ → signaling hotspots at the plasma membrane

Monomeric GTPase

• Arf proteins- COP1 and clathrin at Golgi membrane

• Sar1- COP11 at ER membrane

  • Sar1-GDP in cytosol, Sar-GEF swaps GDP for GTP

  • Sar1-GTP bound to dono ER membrane

  • Sec 23 and 24 bind to Sar1-GTP

  • Cargo receptor and cargo bind to Sec24

  • Sec 13/31 coat Sec 23/24

  • Vesicle buds

Rab Proteins

• Transport vesicles have markers that are complementary to target membrane

• Rab proteins mean specificity

• Step 1:

  • Rab proteins and rab effectors tether transport vesicles to target membrane

  • Ensure membrane specificity

• Step 2:

  • SNARE proteins and SNARE regulators drive fusion of lipid bilayers

Transport Vesicle Tethering

• Vesicle in cytosol has cargo, cargo receptor, v-SNARE, and Rab-GTP

• Rab-GTP tethers to Rab effector and is pulled toward membrane

• v-SNARE tethers to trans-SNARE complex on membrane

• Vesicle fuses, Rab-GTP hydrolyzes and is released with GDI (Rab recycler)

• v-SNARE disconnects

SNARES

• v-SNARES on vesicle

• t-SNARES on target

• v and t-SNARES are attached, water molecules are in between

• Water is pushed out and outer membrane fuses

• Once outer membrane is fused, inner membrane begins fusion

Transport from ER to Golgi using COPII

• Vesicle has cargo, cargo receptor, Sar-1 GTP, adaptor proteins, and outer COPII proteins

• In the ER lumen, resident ER protein, chaperones bounds to misfolded proteins

COPI Retrograde Transport from cis-Golgi to ER

• ER resident proteins, KDEL allows it to be retrieved

  • Help fold proteins

  • Quality control

  • Maintain ER environment

• Secretory protein, passing through on its way to golgi

  • Moved to Golgi for further modification

  • No KDEL, they will move forward

• The Golgi pH is more acidic

  • KDEL binds to KDEL tagged proteins in Golgi tightly

• Higher pH in ER

  • KDEL receptor releases proteins

• On/off switch of KDEL grab and release mechanism due to pH

Golgi Movement

• Golgi cisternae (folds) contain different enzymes

• cis-Golgi- Facing ER membrane

• trans-Golgi- Away from membrane

• Cisternal maturation

  • Vesicles come from ER and create a vesicular tubular cluster

  • This cluster is added to Golgi as a cisterna

  • The further trans-Golgi cisterna is pushed out

  • Enzymes are moved and replaced

• Vesicle transport model

  • Vesicles move between cisterna

Glycosylation in the Golgi

• Glycosylation- Addition of sugars to proteins or lipids

• N-linked glycosylation

  • Sugars attached to the nitrogen on asparagine

• O-linked glycosylation

  • Sugars attach to the oxygen on serine or threonine

• Proteins move through cis → medial → trans Golgi cisternae

• Each compartment contains specific enzymes that modify glycan

• Golgi is the main site for O-linked glycosylation initiation and extension

Movement from Cis → Medial → Trans Golgi for Glycan

• Cis‑Golgi:

  • Phosphorylation of oligosaccharides on lysosomal proteins (creates the mannose‑6‑phosphate tag).

  • Initial mannose removal.

• Medial Golgi:

  • Further mannose trimming.

  • Addition of N‑acetylglucosamine (GlcNAc).

• Trans Golgi:

  • Addition of galactose (Gal) and N‑acetylneuraminic acid (NANA/sialic acid).

  • Sulfation of tyrosines and carbohydrates.

Movement from trans-Golgi to Lysosomes

• vis mannose-6-phosphate tagging in cis-Golgi

• M6P-tagged enzymes bind to M69 receptor in the trans-Golgi

• Moves to endosome

• Late endosome pH drops and M6P receptors release cargo

• Lysosome Acid Hydrolases:

  • Nucleases

  • Proteases

  • Glycosidases

  • Lipases

  • Phosphatases

  • Sulfatases

  • Phospholipases

• Proton pump maintains pH gradient

Lysosome Maturation

• Late endosome contains intraluminal vesicles and inactive hydrolases

• Endolysosome: Fusion of late endosome with a lysosome

  • Mixes cargo, hydrolases, membrane proteins

• Lysosome with acid hydrolases form

• It can regenerate or continue as a lysosome

Endocytosis in Lysosome

• Membrane forms a small endocytic vesicle

• Vesicle fuses with early endosome

• Early to late endosome to lysosome

• Turnover and regulation of membrane components

Phagocytosis in Lysosome

• Membrane form a phagosome

• Phagosome fuses with lysosome to form phagolysosome

• Acid hydrolases digest material

• Immune defense and clearing large debris

Macropinocytosis in Lysosomes

• Membrane ruffles and folds back and form macropinosome

• Macropinosome matures and fuses with endosomes to form lysosome

• Cancer cells use this to scavenge nutrients

Autophagy

• Double membrane structure form autophagosome

  • Engulfs cytosol and organelles

• Autophagosome fuses with a lysosome to form autolysosome

• Contents degraded and recycled

• Quality control, stress response, starvation survival

• Non-selective

  • During starvation

  • Bulk cytosol engulfed

  • Nutrient recycling

• Selective

  • Targets mitochondria, peroxisomes, pathogens

Acid Hydrolases to Endosomes

• Phosphate group added in cis-Golgi

• Mannose-6-phosphate developed in medial Golgi

• m6P receptor bind M6P-tagged hydrolases

  • Cluster into vesicles coated with clathrin and AP1

• Vesicles bud off and fuse with early endosome

• Hydrolases detach due to acidic environment

• Phosphatase removes phosphate and tag is no more

• M6P receptors are packaged into vesicles coated with retromer and reused

Leaving the Golgi

• Signal-Mediated Diversion to Lysosomes (vis endosomes)

• Signal-Mediated Diversion to Secretory Vesicles (Regulated Secretion)

• Constitutive Secretory Pathway (Default)

Signal-Mediated Diversion to Lysosomes

• Lysosomal hydrolases receive an M6P tag in the Golgi

• M6P receptors in the TGN bind these tagged enzymes.

• They are packaged into clathrin‑coated vesicles.

• Vesicles fuse with early endosomes.

• Acidic pH causes hydrolases to release.

• Hydrolases eventually reach the lysosome.

• Highly selective and signal dependent

Signal-Mediated Diversion to Vesicles

• Certain proteins (e.g., hormones, neurotransmitters, digestive enzymes) are sorted into dense‑core secretory vesicles.

• These vesicles do not fuse with the plasma membrane immediately.

• They wait for a signal such as:

  • Ca²⁺ influx

  • Hormonal stimulation

  • Neural activation

• Upon stimulation, vesicles fuse with the membrane and release their contents.

• Controlled, stimulus-dependent release

Constitutive Secretory Pathway (Default)

• Proteins that lack specific sorting signals automatically enter this pathway.

• Vesicles bud from the TGN and move directly to the plasma membrane.

• They fuse continuously and release cargo outside the cell.

• “Always on pathway”

Constitutive Secretory Pathways

• Always on, unregulated

• Vesicles bud from trans-Golgi network

• Travel directly to the plasma membrane

• Vesicle fuse immediately

• Cargo released outside cell or inserted into membrane

Regulated Secretory Pathway

• Signal required: Typically Ca2+, hormones, neurotransmitters

• To plasma membrane

• Proteins destined for regulated secretion are sorted into dense‑core secretory vesicles in the TGN.

• These vesicles do not fuse with the plasma membrane right away.

• They wait in the cytosol until a signal arrives.

• A stimulus (like Ca²⁺ influx) triggers regulated membrane fusion.

• Vesicles release their contents in a rapid, controlled burst.

• Ex. Nerves

Direct Sorting in the Trans‑Golgi Network (TGN)

• The TGN recognizes sorting signals on proteins.

• It packages them into apical vesicles or basolateral vesicles.

• Vesicles travel straight to their correct membrane domain.

• Tight junctions prevent mixing once they arrive.

• Fast

• Sorting signals determine destination

  • GPI-anchored → apical

Indirect Sorting via Early Endosomes

• TGN sends proteins to the basolateral membrane (default route).

• Proteins are internalized into basolateral early endosomes.

• Endosomes sort proteins:

  • Some return to basolateral membrane

  • Some are sent to the apical membrane

• Vesicles deliver sorted cargo to the correct domain.

• Two-step process

• Dynamic regulation

Endocytic Pathway

• Internalizing molecules from plasma membrane

• Receptor-ligand complexes

• Nutrient uptake

• Cell debris/ ECM

• Regulating plasma membrane composition

• Balancing exocytosis, homeostasis

Endocytic Pathway Workflow

• Material internalized from plasma membrane

  • Starting point for receptor downregulation and membrane recycling

• Early endosome

  • Cargo is sorted into different fates.

  • pH drops slightly (~6), causing some receptors to release their ligands.

  • Some receptors are recycled back to the membrane.

  • Others are sent deeper into the pathway for degradation.

• Recycling endosome

  • Sends material back to the plasma membrane

• Multivesicular body

  • Early endosome matures

  • Contains intralumenal vesicles

  • Can’t return back to membrane

• Late endosome

  • pH drops further (~5.5).

  • Cargo is prepared for degradation.

  • Hydrolases from the Golgi arrive via M6P‑mediated targeting.

  • Membrane proteins are sorted into intralumenal vesicles.

• Endolysosome

  • Hydrolases mix with cargo.

  • Digestion begins.

  • Membranes and proteins are broken down.

• Lysosome

  • Fully mature, acidic compartment

• TGN

  • Sends acid hydrolases to endosome using the M6P pathway

• Microtubule mediated transport

  • Move along microtubules using motor proteins

Clathrin in Endocytosis

• Pinocytosis (non-specific)

  • Takes in extracellular fluid

  • Continuous

  • Clathrin-independent

  • Sometimes uses caveolae

• Receptor-mediated

  • Highly selective

  • Clathrin +AP2

LDL as Receptor-Mediated Endocytosis

• LDL binds to LDL receptors on plasma membrane

• Clathrin-coated vesicles form

• Clathrin coat removed

• Fusion with early endosome

• Receptor recycling

• LDL moves to late endosomes

• Fusion with lysosome forms endolysosome

• Free cholesterol moves out

Making Intraluminal Vesicles

• Ligand-bounds receptor enters early endosome

• Add a ubiquitin tag

  • Marks for degradation

• ESCRT recognizes ubiquitin tag

• Inward budding, receptor ends up inside a small vesicle

• Receptor is sequestered and signaling stops

• Fuses with lysosome and degrades

Energetics Overview

• Cells must make and consume ATP

• Mitochondria- Oxidative phosphorylation

• Chloroplasts- Photosynthesis

• Major energy currencies- GTP, NAD+, NADH, FAD, FADH2

Cellular Respiration

• Start with 1 glucose and 2 ATP → 10 linear reactions → 2 pyruvate +4 ATP +2NADH

• Pyruvate processing creates Acetyl CoA

• Citric acid cycle releases CO2 and CoA

• NADH is hydrolyzed'

• 2e- go down electron transport chain

• ADP and Pi are turned into ATP and H2O is created

Glycolysis

• Net synthesis of 2 ATP and 2 NADH

• 1 molecule of glucose takes up 2 ATP to create fructose 1,6-bisphophate

• Fructose 1,6-bisphophate is cleaved into 2 3-carbon sugars

  • Glyceraldehyde 3-phosphate

• Substrate level phosphorylation

  • 2 ATP and 1 NADH out on each sugar

• 2 molecules of pyruvate

Steps 6 and 7 of Glycolysis

• Oxidation of aldehyde is favorable and formation of high-energy P unfavorable

• Oxidation of aldehyde to a carboxylic acid releases energy

• Energy is captured in the activated carriers ATP and NADH

Glycolysis Regulation

• Glucose turns into fructose 6-phosphate

• High ATP inhibits fructose 6-phosphate to slow reaction rate of glycolysis

  • Allosteric

Anaerobic Fermentation

• Occurs without O₂

• Uses fermentation to regenerate NAD⁺

• Net yield: 2 ATP per glucose

• Produces lactate (animals) or ethanol + CO₂ (yeast)

• Enzyme: lactate dehydrogenase (animals), pyruvate decarboxylase + alcohol dehydrogenase (yeast)

• Regulation: triggered when ETC cannot oxidize NADH

• Clinical: lactic acidosis during hypoxia

  • “Must replenish NAD⁺ or glycolysis will stop!”

Aerobic Respiration

• Requires O₂

• Pyruvate → Acetyl‑CoA → Citric Acid Cycle

• Produces NADH, FADH₂, CO₂

• ETC generates proton motive force

• ATP synthase produces ~30–32 ATP per glucose

• Location: mitochondria

• Regulation: depends on oxygen availability & ADP levels

  • “Input: NADH, FADH₂ and O₂… Output: H₂O and H⁺”

Lactic Acid Fermentation

• Pyruvate → Lactate

• Regenerates NAD⁺

• Occurs in muscle cells

• Enzyme: lactate dehydrogenase

• No CO₂ released

• Purpose: maintain glycolysis during O₂ shortage

  • “Fermentation leading to excretion of lactate”

Alcohol Fermentation

• Pyruvate → Acetaldehyde → Ethanol

• Releases CO₂

• Regenerates NAD⁺

• Occurs in yeast

• Enzymes: pyruvate decarboxylase, alcohol dehydrogenase

• “Fermentation leading to excretion of ethanol and CO₂”

Pyruvate Processing

• Pyruvate + NAD⁺ + CoA → Acetyl‑CoA

• Produces 1 NADH + 1 CO₂ per pyruvate

• Occurs in mitochondrial matrix

• Enzyme: pyruvate dehydrogenase

• Regulation: inhibited by high ATP, NADH, acetyl‑CoA

  • “Input: Pyruvate, NAD⁺, Coenzyme A… Output: Acetyl‑CoA, 1 NADH, 1 CO₂”

Citric Acid Cycle

• Inputs: Acetyl‑CoA, NAD⁺, FAD

• Outputs per turn: 3 NADH, 1 FADH₂, 1 GTP, 2 CO₂

• Occurs in mitochondrial matrix

• Key enzymes: citrate synthase, isocitrate dehydrogenase, α‑ketoglutarate dehydrogenase

• Regulation: inhibited by ATP, NADH

  • “3 NADH, 1 FADH₂, 1 GTP and 2 CO₂”

Electron Transport Chain (ETC)

• Uses NADH & FADH₂

• Pumps H⁺ into intermembrane space

• Requires O₂ as final electron acceptor

• Produces H₂O

• Complexes I–IV

• Mobile carriers: CoQ, cytochrome c

  • “Input: NADH, FADH₂ and O₂… Output: H₂O and H⁺”

Proton Motive Force (PMF)

• Combination of pH gradient + membrane potential

• Drives ATP synthesis

• Powers pyruvate import, phosphate import, ADP/ATP exchange

  • “Proton‑motive force due to pH gradient… due to membrane potential”

ATP Synthase

• Uses H⁺ flow to rotate rotor

• Conformational changes produce ATP

• Located in inner mitochondrial membrane

• F₀: proton channel

• F₁: catalytic head

  • “H⁺ flow makes rotor stalk rotate… Bind ADP and Pi, change conformation to make ATP”