Exam 2 (Ch.8, 7, 2, 9)

What is membrane trafficking?

• The movement of proteins, lipids, and other materials between different compartments of a cell

  • Cells have diff organelles that perform specific functions

  • Proteins & lipids must reach correct organelle to function

  • Cell uses vesicles to transport many of these materials

What is the Endomembrane System?

• A group of membranes and organelles that work together to:

  • Produce, modify, and transport lipids & proteins

  • Secrete substances outside the cell (exocytosis)

  • Bring substances into the cell (endocytosis)

• Important organelles

  • Endoplasmic reticulum (ER)

  • Golgi

  • Endosomes

  • Lysosomes

Endoplasmic reticulum (ER) function

Protein & lipid production

Golgi apparatus function

Protein modification and sorting

Endosomes function

Sort internalized materials

Lysosomes function

Digest and recycle materials

What are vesicles?

• A small membrane-bound sac that transports material (protein, lipids, etc) between organelles/compartments

• Form by budding from one membrane

• Deliver materials by fusing w/ another membrane

How does vesicle transport work?

• 1) A protein is synthesized in ER

• 2) Portion of the ER membrane buds outward

• 3) Vesicle containing the protein separates from ER

• 4) Vesicle travels to Golgi

• 5) Vesicle fuses w/ Golgi membrane

• 6) Protein is delivered to Golgi

Soluble proteins vs. Membrane proteins

• Soluble proteins: Located inside vesicle & released into recipient organelle’s interior after fusion

• Membrane proteins: Embedded in vesicle membrane and transferred into recipient organelle’s membrane when fusion occurs

Why do vesicles fuse/go through Golgi?

• To deliver proteins that either need to function in Golgi or need further modifications before reaching their final destination

• 1) Golgi req proteins to perform its function

  • some proteins transported from ER are meant to stay in Golgi

• 2) Proteins could also need further modifications that take place in Golgi

  • Proteins synthesized in rough ER may not be fully processed or ready to function, so vesicle transports these proteins from ER to Golgi to perform additional modifications

Directions of vesicle transport/movement

• Anterograde: Moves material forward through secretory pathway

  • ER → Golgi

  • Golgi → plasma membrane

• Retrograde: Moves material backward or returns/recycles material

  • Golgi → ER

  • Plasma membrane → endosome

  • Allows proteins that belong in ER but accidentally travel to Golgi to return to ER

• Why do we need both directions?

  • Proteins must move forward to reach their destinations, but certain proteins & membrane components need to be returned so organelles maintain their proper functions

How do scientists study membrane trafficking?

• Pulse-chase autoradiography: determines where newly synthesized proteins travel inside the cell

  • Conclusion: proteins move through secretory pathway from ER to Golgi & then toward plasma membrane

• GFP fusion proteins: allows scientists to directly observe protein movement w/out radioactive labeling

  • Conclusion: ER → Golgi

• Cell-free systems

  • Conclusion: Proteins synthesized by ER-bound ribosomes can enter ER lumen

• Mutant screens: scientists mutate genes to determine which proteins are required for membrane trafficking

  • If a cell produces many vesicles but they cannot fuse w/ their destination, the vesicles accumulate → tells scientists that the mutated protein normally helps vesicles fuse

What is secretion/exocytosis?

• Process of releasing large molecules, such as proteins, from inside the cell to outside the cell

• Occurs through secretory pathway, which transports proteins through diff organelles before they are released

• Main secretory pathway:

  • Rough ER → Golgi → secretory vesicle → plasma membrane → outside cell

• First stop: Endoplasmic Reticulum (ER)

Endoplasmic Reticulum (ER)

• The 1st organelle involved in secretory pathway

• 3 main functions: Synthesizes proteins, begins glycosylation, & performs quality control

  • 1) Protein/lipid synthesis

    • Rough ER: synthesizes proteins that will be secreted, inserted into membranes, or transported within endomembrane system

      • proteins synthesized in RER can undergo modifications before being transported to Golgi

    • Smooth ER: synthesizes lipids

  • 2) Protein glycosylation: modification of proteins by adding several oligosaccharides → proteins become glycoproteins

    • Helps proteins fold properly

    • Important for protein quality control

    • Helps proteins function correctly

  • 3) Protein quality control

    • ER checks whether newly synthesized proteins are folded correctly

      • Correctly folded proteins → can leave ER and travel to Golgi

      • Incorrectly folded proteins → retains in ER for further folding attempts

        • Proteins that can’t fold correctly may be sent for degradation

What is the secretory pathway?

• Transports proteins from rough ER through Golgi to their final destinations aka transports proteins through endomembrane system

  • some proteins are secreted outside the cell, while others become part of the plasma membrane or are delivered by lysosomes

• The process by which proteins are synthesized in rough ER, transported to Golgi for modification and sorting, and then delivered to their final destination

• Main pathway:

  • ER → Transport vesicle → Golgi → Transport vesicle → Final destination/plasma membrane

  • Proteins may be delivered to:

    • plasma membrane (*major pathway) → membrane proteins

    • extracellular environment

    • lysosomes → digestive enzymes

    • other endomembrane compartments

Rough ER

• Has ribosomes attached to its surface

• Responsible for synthesizing proteins destined for secretion, membranes, and endomembrane system

• Located near nuclear envelope

Smooth ER

• Doesn’t have ribosomes attached

• Synthesizes lipids & steroid hormones

• Participates in detoxification

• Stores Ca²⁺ ions

Free ribosomes

• Located in cytoplasm

• Produce proteins that may function in cytoplasm or be imported into organelles such as the nucleus & mitochondria

ER-bound ribosomes

• Attached to rough ER during translation

• Produce proteins that enter endomembrane system, be secreted from the cell, or become membrane proteins

• Go to these destinations:

  • Endomembrane system

    • ER

    • Golgi

    • Vesicles

    • Plant vacuoles

  • Secretion

  • Become integral membrane proteins (TM proteins)

How does a soluble protein know to go to ER?

• They contain an ER signal sequence (6-15 hydrophobic amino acids at protein N-term)

  • acts like zip code directing protein to ER

• Signal sequence is bound by Signal Recognition Particle (SRP)

Signal Recognition Particle (SRP)

• A molecule that recognizes the ER signal sequence on a growing protein

• Steps

  • 1) Translation begins on a free ribosome

  • 2) ER signal sequence emerges from ribosome

  • 3) SRP-GTP recognizes & binds the signal sequence

  • 4) SRP temporarily pauses translation

    • Pauses translation so ribosome can be directed to ER before protein continues being synthesized

  • 5) SRP brings the ribosome to the ER membrane

  • 6) SRP binds to an SRP receptor

  • 7) The ribosome becomes positioned near a translocon

    • Translocon: a protein channel in ER membrane

  • 8)GTP hydrolysis helps release SRP which enters translocon (SRP-GTP → SRP GDP)

  • 9) Translation resumes, & the growing protein enters the ER

What is a translocon?

• A protein channel in ER membrane

  • Allows growing proteins to enter ER lumen

  • Can open to allow the protein to pass through

  • Can also release hydrophobic transmembrane segments into lipid bilayer

Cotranslation translocation

• Process where a protein enters ER while it’s being synthesized (protein doesn’t have to be completely synthesized before entering ER)

• Translation & translocation occur simultaneously

  • Translation: ribosome makes a protein by joining amino acids together

  • Translocation: growing protein moves into ER through protein channel called translocon

• Purpose: Allows a growing, mostly unfolded protein to enter ER through translocon → makes transport easier b/c unfolded proteins can pass through the narrow channel

  • THEN once inside ER, chaperones help protein fold into its functional 3D shape

• Steps:

• 1) Ribosome begins translation in cytosol

• 2) SRP recognizes signal sequence & pauses translation

• 3) SRP directs ribosome to SRP receptor on rough ER

• 4) Ribosome attaches to translocon, & SRP is released

• 5) Translation resumes → growing protein enters ER through translocon (translation & translocation occurring simultaneously)

• 6) BiP & other chaperones help protein fold correctly

• 7) Signal peptide is often removed by signal peptidase

• 8)Once translation finishes, a soluble protein can be released into ER lumen

Start-transfer sequence vs. Stop-transfer sequence

• Start-transfer sequence: A hydrophobic sequence that initiates movement of a protein through the translocon

• Stop-transfer sequence: A hydrophobic sequence that stops movement of the protein into ER lumen & becomes embedded in the membrane

Soluble protein translocation into ER

• After proteins are synthesized by ribosomes, they need to enter ER lumen or become embedded in ER membrane

• This protein enters ER lumen completely

• Steps

  • 1) Protein synthesis begins on ribosome in cytoplasm

  • 2) The protein contains a hydrophobic signal peptide that acts as a start-transfer sequence

    • It directs the protein to the ER & initiates movement through translocon

  • 3) The growing protein passes through translocon into ER lumen

  • 4) Signal peptidase cleaves the signal peptide during translocatioin

  • 5) The mature protein is released into ER lumen & folds

Single-Pass Transmembrane Protein: Method 1

• Purpose: Produce a protein that crosses the ER membrane once

• Requires both a start-transfer & a stop-transfer sequence

• Steps:

  • 1) Protein synthesis begins in cytoplasm

  • 2) Protein contains an N-terminal signal peptide that acts as a start-transfer sequence

    • Initiates movement through translocon into ER lumen

  • 3) A second hydrophobic sequence called the stop-transfer sequence reaches the translocon

    • Stops further movement of the protein into ER lumen

  • 4) The stop-transfer sequence is released sideways from translocon into lipid bilayer

    • It becomes the transmembrane segment that anchors the protein

  • 5) Signal peptidase cleaves the original signal peptide

  • 6) Translation finishes, leaving the protein embedded in ER membrane

Single-Pass Transmembrane Protein: Method 1 Final Result

• N-terminus → ER lumen

• C-terminus → Cytoplasm

• Stop-transfer sequence → Embedded in ER membrane

Single-Pass Transmembrane Protein: Method 2

• Purpose: Produce a single-pass protein using an internal ER recognition sequence

• Uses an internal start-transfer sequence that isn’t removed

• Steps:

  • 1) Protein synthesis begins in cytoplasm

  • 2) The protein contains an internal hydrophobic signal sequence instead of an N-terminal signal peptide

  • 3) SRP recognizes the internal signal sequence & directs the ribosome to ER

  • 4) The internal signal sequence inserts into translocon and acts as both a start-transfer sequence & a membrane anchor

    • N-terminal portion remains in cytoplasm

    • C-terminal portion enters ER lumen

  • 5) Internal signal sequence remains in embedded in ER membrane and is not cleaved

Single-Pass Transmembrane Protein: Method 2 Final Result

• N-terminus → Cytoplasm

• C-terminus → ER lumen

• Internal signal sequence → Embedded in ER membrane

Single-Pass Transmembrane Protein: Method 3

• Purpose: Produce a single-pass protein w/ the opposite orientation from Method 2

• Uses internal signal sequences like Method 2, but the protein inserts in opposite orientation

• Steps:

  • 1) Protein synthesis begins in cytoplasm

  • 2) The protein contains an internal hydrophobic ER recognition sequence

  • 3) Internal sequence directs insertion into translocon

  • 4) Internal sequence inserts in the opposite orientation compared w/ Method 2

    • N-terminal portion enters ER lumen

    • C-terminal portion remains in cytoplasm

  • 5) Internal sequence remains embedded in ER membrane and is not cleaved

Single-Pass Transmembrane Protein: Method 3 Final Result

• N-terminus → ER lumen

• C-terminus → Cytoplasm

• Internal signal sequence → Embedded in ER membrane

Double-Pass & Multipass Transmembrane Proteins

• Double-pass proteins: Cross ER membrane twice

• Multipass proteins: Cross ER membrane multiple times

• These proteins contain multiple hydrophobic sequence that directs their insertion into membrane

• Steps of multipass transmembrane proteins

  • 1) A start-transfer sequence initiates movement of protein into ER

  • 2) A stop-transfer sequence stops movement into ER lumen

  • 3) Another start-transfer sequence can initiate translocation again to enter ER lumen again

  • 4) Another stop-transfer sequence can stop translocation and stop proteins from entering again

  • 5) Repeated start-transfer and stop-transfer sequences creates multiple membrane-spanning regions

  • Final result: protein crosses ER membrane multiple times

Rough ER…

• Makes transmembrane proteins

• Determines their orientation in membrane

• Prepares them for their functions at plasma membrane

How does rough ER make transmembrane proteins?

• TM proteins = proteins that span across phospholipid bilayer

  • synthesized by ribosomes attached to rough ER

  • contain 1 or more hydrophobic transmembrane segments that become embedded in membrane

  • some become receptors on plasma membrane

• Steps:

  • 1) A ribosome begins making a protein, which is directed to rough ER

  • 2) The growing protein enters translocon in ER membrane

  • 3) When hydrophobic transmembrane segment reaches translocon, it can move sideways into phospholipid bilayer

  • 4) The hydrophobic segment stays embedded in membrane, anchoring the protein

  • 5) Depending on its amino acid sequence, diff parts of protein face cytoplasm or ER lumen

What determines orientation of TM proteins?

• The distribution of charged amino acids surrounding hydrophobic TM segment

  • More positively charged amino acids side of TM segment remain facing cytoplasm

  • Opposite side tends to face ER lumen (Note: negatively charged amino acids don’t always have to face ER lumen)

• Two ends:

  • a) N terminus, NH₂ (beginning of protein)

  • b) C-terminus, COOH (end of protein)

• When a protein travels from ER to plasma membrane, its orientation is preserved b/c its orientation is established during synthesis in ER

  • Part facing ER lumen (space inside ER) will eventually face extracellular environment

  • Part facing cytoplasm (an area inside cell) will remain facing cytoplasm

Example of Proteins Having Diff Orientations: LDL Receptor

• LDL: a particle that transports cholesterol & other lipids through blood

• LDL receptor: a transmembrane protein that binds LDL & helps bring it into cell through receptor-mediated endocytosis

• LDL receptor orientation:

  • N-terminus has negatively charged aa close by: faces ER lumen during synthesis & eventually faces outside cell

    • Contains region that binds LDL

  • C-terminus has positively charged aa close by: faces cytoplasm

    • Contains signals needed to recruit machinery for endocytosis

  • TM domain: anchors receptor in membrane

• LDL receptor needs its N-terminus outside to bind LDL & its C-terminus inside to help bring LDL into cell

Example of Proteins Having Diff Orientations: Transferrin receptor

• Transferrin (TF): A protein that carries iron in blood

• Transferrin receptor: a transmembrane protein that binds iron-loaded transferrin & helps bring it into cell through receptor-mediated endocytosis

• Transferrin receptor orientation:

  • N-terminus has positively charged aa close by: faces cytoplasm

    • Contains signal needed for endocytosis

  • C-terminus has negatively charged aa close by: faces ER lumen during synthesis & eventually faces outside the cell

    • Binds transferrin, allowing receptor to capture iron-loaded transferrin

  • TM domain: anchors receptor in membrane

• Transferrin receptor has opposite N/C orientation from LDL receptor, but both perform receptor-mediated endocytosis

How are multiple TM segments inserted?

• They cross the membrane several times

• The 1st TM segment helps establish orientation

• Subsequent TM segments alternate their directions across the membrane

What is protein glycosylation?

• The addition of carbohydrate chains (oligosaccharides) to proteins → glycoproteins

• Why is it important?

  • Helps protein fold properly

  • Supports cell-to-cell interactions

  • Contributes to protein function

• 2 types:

  • a) N-linked: oligosaccharide attached to asparagine (Asn)

    • Begins in ER, modified further in Golgi

  • b) O-linked: oligosaccharide attached to serine (Ser) or threonine (Thr)

    • Occurs in Golgi, modified in Golgi

How does N-linked glycosylation happen in rough ER?

• 1) Glycosyl transferase (enzyme) helps assemble the branched oligosaccharide containing 3 glucose molecules at its tip → adds sugars

• 2) Branched oligosaccharide (sugar chain) containing 3 glucose molecules at its tip is attached to asparagine (Asn)

  • This occurs co-transitionally (while translation is still happening)

  • Transfer occurs on ER lumen side

• 3) The sugar chain is modified in ER

  • Glucose molecules are removed from sugar chain by enzymes

  • These modifications help the protein fold correctly

• 4) Protein is transported from ER to Golgi in transport vesicle

  • In Golgi, additional sugars may be added or removed to further modify the protein

• 5) Glycosylated protein travels to final destination

  • Can become a plasma membrane TM protein or a secreted protein

Protein quality control in ER

• ER checks whether newly synthesized proteins have folded correctly

• Important proteins:

  • BiP: chaperone that helps proteins fold

  • Calnexin: chaperone involved in glycoprotein folding

  • Calreticulin: another ER chaperone

  • Glucosidases: removes glucose residues from glycoproteins

• Steps:

  • 1) A newly synthesized protein enters ER

  • 2) Chaperones help the protein fold

  • 3) ER checks whether the protein is folded correctly

  • 4) If correctly folded, protein can leave ER

  • 5) If incorrectly folded: protein may undergo another folding cycle

  • 6) If protein cannot fold correctly, it may be sent to cytoplasmic proteasome for degradation

What happens when misfolded proteins accumulate in ER?

• Response: Unfolded Protein Response (UPR) → a cellular response triggered by accumulation of misfolded proteins in ER

• UPR sensors: proteins located in ER membrane that detect problems w/ protein folding

• Under normal conditions:

  • BiP charperone not busy and is available b/c few misfolded proteins in ER

  • BiP binds UPR sensors & keeps them inactive → UPR is OFF

• When misfolded proteins accumulate:

  • BiP is recruited to help these proteins fold & is busy

  • BiP dissociates from UPR sensors which makes them become active

  • UPR is ON and initiate signaling pathways that help cell respond to ER stress

    • 2 responses:

      • 1) Phosphorylate translation factor elF2α to stop translation/production of more proteins

      • 2) Part of cytoplasm is cut, released, & acts as transcription factor that needs to go to nucleus to make more charperone proteins to reinforce proper protein folding

What is the Golgi apparatus?

• Responsible for:

  • Modifying proteins

  • Processing proteins

  • Sorting proteins (secretion, PM, lysosomes)

  • Packaging proteins for transport

• Regions of Golgi:

  • Cis-Golgi Network

  • Cis-Golgi

  • Medial-Golgi

  • Trans-Golgi

  • Trans-Golgi network

Regions of Golgi: cis-Golgi network

• Receives vesicles from ER

• Closest to nucleus/ER

Regions of Golgi: trans-Golgi network

• Sorts proteins/vesicles to different destinations

• Farthest from nucleus/ER

Regions of Golgi: cis, medial, trans-Golgi

• Protein processing

  • cis: early protein processing

  • medial: further modification

  • trans: later modification

Protein processing in Golgi: Proteolysis

The cleavage of a protein to make shorter functional proteins

• Why? → Some proteins become functional only after being cleaved into smaller pieces

• Ex. POMC

  • One gene produces one transcript

  • The transcript produces one polypeptide

  • The polypeptide can be processed into multiple peptide hormones

Protein processing in Golgi: Glycosylation

Adding or modifying sugar chains attached to proteins

• Important for:

  • Protein folding & stability

  • Cell recognition & cell-cell interactions

  • Protein trafficking & function

• 2 types:

  • N-linked: begins at rough ER, modified in Golgi

  • O-linked: Begins in Golgi, modified in Golgi

Why are Golgi regions important?

• Different regions of Golgi contain different enzymes that carry out different modifications → Golgi functions are regionalized

• Proteins progress through these regions:

• 1) Cis-Golgi: early modifications, including removal of mannose residues

• 2) Medial Golgi: further mannose removal & addition of other sugars

• 3) Trans-Golgi: later modifications, including addition of galactose & sialic acid in many glycoproteins

A Glycosylation Pattern: Enzyme α-Mannosidase I

• Located in cis-Golgi

• Function: Removes mannose residues

A Glycosylation Pattern: Enzyme GlcNAc-transferase I

• Located in medial-Golgi

• Function: Adds N-acetylglucosamine (GlcNAc)

A Glycosylation Pattern: Enzyme α-Mannosidase II

• Located in medial-Golgi

• Function: Removes additional mannose residues

A Glycolsylation Pattern: Enzyme GlcNAc-transferase II

• Located in medial-Golgi

• Function: Adds another N-acetylglucosamine

A Glycosylation Pattern: Enzyme Fucosyltransferase

• Located in medial-Golgi/trans-Golgi

• Function: Adds frucose

A Glycosylation Pattern: Enzyme Galactosyltransferase

• Located in trans-Golgi

• Function: Adds galactose

A Glycosylation Pattern: Enzyme Sialyltransferase

• Located in trans-Golgi

• Function: Adds sialic acid

How do proteins move through Golgi?

• 2 Models:

• a) Vesicular transport model

  • Vesicles carry cargo from one Golgi cisterna to the next

  • Golgi enzymes remain in their compartments

• b) Cisternal maturation model

  • Cargo remains inside a cisterna as it matures

  • Golgi enzymes move backward through retrograde vesicles

• Note: current evidence supports cisternal maturation model

What is exocytosis?

• The release of substances outside cell through vesicle fusion w/ plasma membrane

• 2 Types:

  • a) Constitutive secretion

    • Not regulated b/c cells know its molecules to throw away (excreted products, damaged parts) → doesn’t require a specific release signal

    • Occurs continuously

  • b) Regulated secretion

    • Is regulated → requires a signal

    • i.e. secretion of neurotransmitters

What is synaptic vesicle exocytosis?

• An example of regulated secretion

• Steps

  • 1) Synaptic vesicles contain neurotransmitters

  • 2) Vesicles dock near plasma membrane since they need to fuse w/ it and waits for signal to fuse

  • 3) An action potential arrives

  • 4) Voltage-gated Ca²⁺ channels open

  • 5) Ca²⁺ enters the cell

  • 6) The signal triggers vesicle fusion

  • 7) Neurotransmitters are released

What are lysosomes?

• Digestive organelles

  • Contain acid hydrolases

  • Have acidic interior (~ pH 4.6)

  • Break down old organelles & other materials

  • Participate in autophagy

  • Help certain immune cells digest bacteria

• Is a secretory pathway destination

How do lysosomal enzymes reach lysosomes?

• They follow secretory pathway:

  • Synthesized in ER → Modified in Golgi → Endosomes → Lysosomes

• Why? B/c lysosomal enzymes are proteins that enter endomembrane system, so they must be synthesized, processed, sorted, & delivered to lysosomes

• Lysosomes are part of antegrade movement & endocytosis movement

• Lysosomal enzymes are translated/synthesized in ER-bound ribosomes

Where are proteins going through endomembrane trafficking system synthesized?

In the rough ER

How are proteins transported through the secretory pathway?

The cell needs several types of proteins to go from Rough ER → Golgi → Transport vesicle → Final destination

• Coat proteins: Help membrane bend to form a vesicle & select which cargo goes inside

• Small GTPases: Regulate vesicle formation & help coat proteins attach/targetting

• Motor proteins: Move vesicles along cytoskeleton toward their destinations

• Rab proteins: Help vesicles recognize & target specific compartments

  • are small GTPases

• SNAREs and SNAPs: Help regulate vesicle docking & fusion → brings vesicle & target membranes together so they can fuse

What are the main functions of coat proteins?

• 2 major jobs:

  • a) Promote membrane curvature

    • More coat proteins = more curvature → creates vesicle

  • b) Select cargo and specifies movement to where

Types of Coat Proteins: COPII

• Form around vesicles that go ER → Golgi

• Transport type: Anterograde

• Helps proteins leave ER

• 2 Functions:

  • a) Promote membrane curvature to help form vesicles

  • b) Select cargo that needs to leave ER

• Types of cargo it can transport:

  • Soluble proteins: proteins located inside ER lumen that need to travel to Golgi

  • Membrane proteins: proteins embedded in ER membrane that need to be transported

  • Cargo receptors: Membrane proteins that bind certain soluble cargo molecules & help package them into COPII vesicles

• Its coat assembly is regulated by a small GTPase called Sar1

How does COPII coat assembly work?

• Sar1: small GTPase that regulates COPII assembly

  • Sar1-GDP = Inactive

  • Sar1-GTP = Active

• Steps:

  • 1) Sar1 is initially inactive, bound to GDP

    • Sar1-GDP = primarily soluble in cytoplasm

    • Not yet recruiting COPII coat proteins

  • 2) GEF activates Sar1

    • GEF in ER membrane promotes release of GDP

    • GTP binds to Sar1 → Sar1 becomes active

  • 3) Sar1-GTP becomes embedded in ER membrane

    • GTP binding causes conformational change in Sar1 which exposes an ampihpathic helix that’s inserts into ER membrane

    • Active Sar1 can now recruit COPII coat proteins

  • 4) Sar1-GTP recruits Sec23 and Sec24

    • Sec23/24 form inner COPII coat

    • Sec24 helps select cargo proteins

    • Cargo receptors help capture soluble cargo

  • 5) Sec13 and Sec31 are recruited

    • Sec13/Sec31 form outer COPII coat

    • Outer coat helps stabilize & shape curved membrane

    • Developing vesicle bud becomes more pronounced

  • 6) COPII vesicle buds from ER

    • Vesicle separates from ER membrane and carries selected cargo toward Golgi

    • Sar1 eventually hydrolyzes GTP to GDP, promoting coat disassembly

    • Uncoated carrier can then undergo targeting & fusion

Types of Coat Proteins: COPI

• Form around vesicles that go: Golgi → ER

  • a) trans-Golgi → cis-Golgi

  • b) cis-Golgi → ER

• Transport type: Retrograde

• Helps return proteins to ER

• Why does the cell need this coat protein?

  • Some ER resident proteins (that contain KDEL) accidentally enter COPII vesicles

  • These proteins reach Golgi even though they belong in ER

  • Cell recognizes them using retrieval signals

    • KDEL: indicates that protein needs to go back to rough ER

      • K= lysine

      • D= Aspartic acid

      • E= Glutamic Acid

      • L = Leucine

  • COPI-mediated transport helps return them to ER

How does COPI return proteins to ER?

1. An ER-resident protein containing KDEL accidentally reaches Golgi

2. A KDEL receptor in Golgi recognizes & binds COPI coat

3. COPI coat machinery is recruited to membrane

4. The membrane bends & forms a COPI-coated vesicle

5. The vesicle transports the protein back toward the ER

6. The vesicle fuses w/ ER membrane, & the protein is released into ER lumen

Types of Coat Proteins: Clathrin

• Helps forms vesicles during endocytosis & during sorting of certain proteins toward lysosomal pathway

• Transport routes:

  • trans-Golgi network → Endosome → Lysosome

  • Plasma membrane → Endosome → Lydosome (sometimes)

• Structure:

  • Forms a 3-legged structure called triskelion

    • each triskelion contains 3 heavy chains & 3 light chains

    • many triskelions assemble into a coat that helps curve the membrane

• Important for endocytosis & cargo transport toward endosomes

What are lysosomal enzymes?

• Organelles that break down & recycle materials inside the cell

• Contain digestive enzymes called acid hydrolases which break down protein, lipids, carbohydrates, and other molecules

• Are synthesized on rough ER and transported through Golgi

• For these enzymes to function in lysosomes, they must be delivered into correct destination

How does the Golgi know which proteins should go to lysosomes instead of being secreted?

• Certain lysosomal enzymes in the Golgi are tagged w/ Mannose-6-phosphate

  • Lysosomal enzymes are glycoproteins, meaning they contain attached carbohydrate chains

  • Mannose = a sugar within those carbohydrate chains

  • Golgi adds a phosphate-containing tag to mannose, producing mannose-6-phosphate which identifies the enzyme as cargo destined for the lysosome

How does the lysosomal enzyme get packaged into a vesicle?

• 1) Mannose 6-phosphate (M6P) receptor binds M6P tag on lysosomal enzyme in trans-Golgi network

• 2) Arf1 (a small GTPase) binds GTP & becomes active

  • Active Arf1 associates w/ Golgi membrane & recruits GGA adaptor proteins

• 3) GGA interacts w/ Arf1-GTP & the cytoplasmic tail of M6P receptor and helps recruit clathrin

• 4) Clathrin proteins assemble around budding membrane → helps form a transport vesicle containing the lysosomal enzyme & its receptor

• 5) The vesicle separates from Golgi membrane and travels toward an endosome

  • Its clathrin coat is removed before vesicle fuses w/ its target memrbane

• 6) Lysosomal enzymes separate from M6P receptors in endosome

• 7) M6P receptors recycle back to trans-Golgi network to bind to other M6P-tagged lysosomal enzymes

• 8)Lysosomal enzymes are delivered to lysosomes

What is an effector for Arf1?

An adaptor protein that recruits the clathrin coat proteins

How do vesicles move?

• Vesicles use motor proteins that move along cytoskeletal tracks

• Motor protein type changes depending on direction of movement:

  • Toward microtubule (+) end → carried by Kinesin

  • Toward microtubule (-) end → carried by Dynein

Vesicle tethering, docking, and fusion

• Tethering

  • Brings a vesicle near its target membrane

  • Tethers can capture vesicles from approx 50-200 nm away

• Docking

  • Positions a vesicle very close to target membrane

  • Prepares vesicle for fusion

  • v-SNAREs (located on vesicle membrane) interact w/ t-SNAREs (located on target membrane)

    • Their interaction helps position & hold vesicle against target membrane

• Fusion

  • SNARE proteins form a tight complex through their alpha-helical regions

    • This pulls the two membranes close enough to promote fusion

  • The vesicle releases its contents into the target compartment

SNARE proteins

• Help w/ vesicle docking & membrane fusion

• v-SNARE and t-SNARE interact to form a SNARE complex

  • v-SNARE: located on vesicle membrane

    • ex. Synaptobrevin

  • t-SNARE: located in target membrane

    • ex. SNAP-25 and Syntaxin

• SNARE complex helps dock the vesicle & pulls the two membranes together to promote fusion

Bacterial neurotoxins that target SNAREs

• Clostridium botulinum and Clostridium tetani = proteases that cleave SNARE proteins

• This prevents synaptic vesicle fusion and disrupts neurotransmitter release

What is protein folding?

• Proteins are made of chains of amino acids called polypeptides

• A polypeptide must fold into a specific 3D shape to function properly

• Correctly folded functional shape = native conformation

• Protein foldings is influenced by interactions between amino acid side chains (R groups)

Why is protein folding complicated? + Folding Pathways

• Scientists don’t completely understand every detail of how proteins fold

• Diff proteins can follow diff folding pathways

• Secondary structures (α-helices and β-sheets) can form at diff stages of folding

• Protein collapse: Unfolded protein becomes more compact as hydrophobic aa side chains tend to move toward inside, away from water

• Two possible folding pathways:

  • Pathway A: Secondary structure forms FIRST

    • Unfolded protein → Secondary structure forms (α-helices and β-sheets forms) → Protein collapses/becomes compact → Native protein

  • Pathway B: Protein collapses FIRST

    • Unfolded protein → Protein collapses/becomes compact → Secondary structure forms (α-helices and β-sheets form) → Native protein

Why do most proteins need help folding?

• Main problem: Hydrophobic regions can aggregate which prevents proteins from reaching their correct native conformation

  • Aggregation: proteins sticking together, often b/c exposed hydrophobic regions interact

• How?

  • Hydrophobic amino acids have non-polar side chains & tend to avoid water

  • During folding, these hydrophobic regions may be temporarily exposed

  • Exposed hydrophobic regions can interact with:

    • Hydrophobic regions of same protein, potentially causing incorrect folding

    • Hydrophobic regions of other proteins, causing proteins to stick together

What are molecular chaperones?

• Proteins that help other proteins fold properly

• They bind to & temporarily shield exposed hydrophobic regions of unfolded or partially folded proteins

  • This prevents unwanted aggregation & allows proper folding to occur

• How do they work?

  • 1) A polypeptide is synthesized by a ribosome

  • 2) Hydrophobic regions of the polypeptide become exposed

  • 3) Molecular chaperones bind to these hydrophobic regions

  • 4) This prevents the polypeptide from aggregating w/ other proteins

  • 5) Chaperones release the polypeptide, allowing it to continue folding into its native conformation

• Note: Chaperones don’t act as exact molds that determine final shape of proteins, but, instead, help proteins fold by preventing inappropriate interactions

Hsp70 Family of Chaperones

• Stands for Heat Shock Protein 70

• Is a family of molecular chaperones

• Can function co-transitionally (while polypeptide is still being synthesized)

• How do they work?

  • 1) A ribosome begins producing a polypeptide

  • 2) Hsp70 binds to exposed hydrophobic regions of the growing polypeptide

    • This prevents aggregation while translation continues

  • 3) The polypeptide is released & can fold into its native structure

Hsp70 and High Temps

• High temps can cause proteins to unfold or become denatured

  • Denaturation: a protein loses its normal 3D structure

• 2 Responses to High Temp

  • a) Promotes misfolding of proteins

  • b) Promotes transcription from chaperone encoding genes → make more chaperones that will try to fold misfolded proteins properly

• This creates process

  • 1) High temp promotes protein misfolding

  • 2) Heat shock promotes transcription of chaperone-encoding genes

  • 3) More chaperone proteins are synthesized

  • 4) These chaperones help protect proteins from aggregation & assist their refolding

What are Chaperonins?

• Another type of specialized molecular chaperone

• Made up of multiple protein subunits

• They form barrel-shaped chambers in which proteins can fold

  • These chambers help isolate unfolded proteins from other proteins that could cause aggregation

• 2 examples:

  • TRiC: a barrel-shaped chaperonin complex

  • GroEL: a bacterial chaperonin belonging to Hsp60 family

GroEL

• Folding chamber

• A chaperonin found in bacteria

• Belongs to Hsp60 family

• Contains 14 polypeptide subunits

• These 14 subunits form 2 stacked rings, each containing 7 subunits

• The rings form chambers where proteins can fold

GroES

• A separate protein complex that acts as a cap for GroEL → cap that closes the chamber

• Binds to one end of GroEL complex

• Helps enclose the unfolded protein inside the folding chamber

How does GroEL and GroES Help Proteins Fold

• 1) GroEL binds an unfolded protein

  • When GroES is not attached, the binding surface inside GroEL is hydrophobic

  • The hydrophobic surface binds exposed hydrophobic regions of an unfolded protein

    • This temporarily holds the protein inside the chamber

  • Why? → Hydrophobic regions of the unfolded protein interact w/ hydrophobic binding surfaces of GroEL

• 2) ATP and GroES bind to GroEL

  • ATP binds to GroEL

  • GroES attaches to GroEL and forms a cap

  • The unfolded protein becomes enclosed inside chamber

• 3) GroEL chamber changes shape

  • When ATP & GroES bind:

    • GroEL undergoes a conformational change

    • Chamber enlarges

    • Chamber’s inner surface becomes hydrophilic

    • Unfolded protein is released from its interactions w/ chamber wall

• 4) Protein folding occurs

  • The protein folds inside enclosed chamber

  • GroES keeps protein separated from other proteins in cell

  • ATP is hydrolyzed to ADP during the folding cycle

• 5) GroES detaches & the protein is released

  • GroEL/GroES complex undergoes additional ATP-dependent changes

  • GroES detaches

  • Protein is released from chamber

  • Protein may be correctly folded or may still be misfolded

  • If protein is not correctly folded, it may undergo another folding cycle

Why is correct protein folding important?

• Proteins must fold correctly to perform their normal cellular functions

• If proteins misfold they …

  • May lose their normal function

  • May expose hydrophobic regions that should be normally buried

  • May aggregate w/ other proteins

• Protein misfolding can contribute to disease

  • Common features of protein folding diseases

    • Conversion of α-helices into β-sheets → indicates that hydrophobic regions aren’t folded properly & interacts w/ other hydrophobic regions which forms aggregates