Cell Biology Chapter 8 Membrane Trafficking

Chapter 8: Membrane Trafficking

• Trafficking 1: Secretory System

  • e.g. hormones and enzymes

  • proteins synthesized in ER, are packaged into vesicles and transported to the Golgi apparatus for further modification and sorting.

    1. proteins start in RER, ribosomes translate mRNA into protein

    2. ER modifies proteins by glycosylation and quality control ensures proper folding

    3. proteins/lipids move from RER → Golgi and undergo further modification

    4. Golgi sorts proteins to final destinations (PM, lysosomes, secretory vesicles) and then they are packaged into vesicles

  • Constitutive secretion: continuous discharge

    • materials synthesized in ER are transported to Golgi → PM

    • maintain and expand PM and produce components for extracellular matrix

  • regulated secretion: secretory vesicles stored in cytoplasm until stimulus triggers release

    • only released to specific signals

Proteins involved in secretory pathway

• Soluble Proteins

  • released into extracellular space via exocytosis

  • e.g. hormones, digestive enzymes, anitbodies

• Intergral membrane proteins

  • embedded in lipid bilayer of organelles or PM

  • e.g. receptors, ion channels, transporters

• Resident proteins of membrane bound organelles

  • soluble proteins that functions inside specific organelles rather than being secreted

  • e.g. lysosomal enzymes (remain in lysosomes to degrade cellular waste)

• Trafficking 2: Endocytosis and Exocytosis

  • Endocytosis:

    • move materials (vesicles) from cell surface to internal components (endosomes, lysosomes.)

    • uptake and transport of external materials through vesicular transport

  • Exocytosis:

    • expel BIG materials, like hormones and neurotransmiters.

    • “secretory pathway”

    • ER → Golgi → PM

      • ER 1st stop: protein/lipid synthesis, protein glycosylation (modification), overall quality control with help of chaperones

    • TM proteins in PM

    • Lysosomes

The Endomembrane System

Involved in

• secretion, endocytosis, includes golgi apparatus, RER, and motor proteins

coordinated unit to transport materials

~ mitochondria, nucleus, and chloroplast NOT part of system

• A group of interconnected membranes and organelles within cells (suspended in cytoplasm of eukaryotic cell) that handle lipid and protein production, modification, and transport.

  • proteins made in ER, processed in Golgi and transported

• Functions include:

  • Exocytosis/secretion

  • Endocytosis

• Inside of the cell:

  • Lipid and Protein production, modification, and transport

Organelles in the Endomembrane System

• Key Organelles:

  • Endoplasmic Reticulum (ER): protein and lipid synthesis and modifications, studded with ribosomes

  • Golgi Apparatus: modify, package, and distribute proteins from the ER. Directs proteins to correct destination (secretion, membrane, lysosome)

  • Endosomes: intermediate vesicles that sort incoming molecules from the PM and deliver them to lysosomes

  • Lysosomes: break down waste material

  • vacuoles: store nutrients and waste products, and help maintain turgor pressure in plant cells.

Membrane-Bound Organelles (Endomembrane System)

• Transport between organelles: vesicles and cargo molecules

• Anterograde: Endoplasmic reticulum → Golgi apparatus → out (exocytosis)

  • Proteins synthesized in RER lumen and excreted

• Retrograde: Golgi → Endoplasmic reticulum → PM → endosomes and lysosomes

  • materials are moved back to ER or Golgi from cell membrane or other organelles (recycling)

Pathways for Transmembrane (TM) Proteins

• Correct routes for TM proteins functioning at the plasma membrane:

  • ER → Golgi → PM

Research Techniques

• • Pulse-chase autoradiography

  • autoradiograohy: visualizes the synthesis and transport of secretory proteins

    • radioactive amino acids track protein movement

    • Pulse: label cells with radioactive amino acids

      1. cells incubated with labeled precursor

      2. label incorporated into newly synthesized proteins

    • Chase: removal of labels to track protein

    • Findings: discovered ER as site of secretory pathway. Determines the movements of newly synthesized materials within the cell

      • ER → Golgi → PM (route for a TM protein)

  • GFP fusion proteins

    • real-time tracking of proteins in LIVE cells

    • no radioactivity needed

    • mammalian cell in culture is infected with virus (VSV)

      • VSVG-GFP (glycoprotein) → ER

    • temp-sensitive mutations = controlled movement

    • when infected, cells become factories for viral protein production = observation of protein trafficking

      • Restrictive temp (40) = VSVG protein cant get out of ER ; this indicates that the proper folding and assembly of the VSVG-GFP protein is temperature-dependent

      • Permissive temp (32) = can get out of the ER and progress to the Golgi apparatus → P<

    • Conclusion: ER → Golgi

  • Cell-free systems

    • study isolated microsomes (endoplasmic reticulum + ribosomes)

      • protein synthesis → protein interior of microsomes

    • Cellular fractionation by centrifugation: allows separation of different organelles based on properties

    • Strip ribosomes from microsomes

      • Protein synthesis → protein in solution

    • Conclusion: ER ribosomes → ER lumen

  • Mutant studies in model organisms like yeast.

    • sec mutant help identify genes controlling vesicle budding/formation and fusion

    • Sec12: Mutations in this gene lead to excessive accumulation of ER membranes, resulting in the inability of vesicles to bud off properly and subsequently fuse with their target membranes.

    • Sec17 = too many vesicles = close to membrane but cant fuse

Endoplasmic Reticulum (ER)

highly dynamic, undergoes continuous reorganization, membrane bound, associated with nuclear envelope → cytoplasm

proteins made in RER have KDEL. Shows protein functions in ER

• RER

  • ribosomes attached on outer surface of membrane

  • sheet like cisternae

  • connected to nuclear envelope. continuous with outer membrane of NE.

  • site of protein synthesis; proteins can undergo glycosylation

  • quality control (protein folding and degradation)

  • intracellular transport

• SER

  • involved in lipid and steroid hormone synthesis

  • lacks ribosomes

  • throughout cytoplasm

  • detoxification of organic compounds like ethanol and barbiturates

    • detoxified compounds are carcinogenic

  • stores Ca2+ in muscle cells

  • release glucose into bloodstream

Integral Membrane Proteins

• enzymes responsible for lipid synthesis have active sites facing the cytosol

• Protein Synthesis in RER

  • synthesize secretory proteins (hormones, enzymes), integral membrane proteins, lysosomal enzymes

  • Protein destinations:

    • ER, golgi, lysosomes, vesicles, plant vacuoles

  • “free” ribosomes don’t go through secretory pathway = protein destination is cytoplasm

  • protein synthesized on “ER bound” ribosomes = destination is the ER, secretions, and lysosomes

    • ER → golgi → PM → secretion

      OR

    • ER → golgi → lysosome

RER: protein synthesis

• Ribosomes attached to RER synthesize:

  • secretory proteins

  • integral membrane proteins

  • lysosomal enzymes

• soluble proteins that are translated on RER-bound ribosomes contain signal sequences that direct ribosomes to RER membrane lumen

  • typically short hydrophobic stretches of amino acids at N-term/AMINO-terminus( ER lumen).

• Signal recognition particle (SRP) binds to signal sequence = temporarily blocks translation

  • SRP contains small GTPase, SRP-GTP that facilitates the targeting of the ribosome

• SRP binds SRP receptor and docks on translocon

• SRP-GTP hydolyzes GTP → SRP-GDP

  • signal released from SRP-GDP and enters translocon = displaces the plug

  • TM proteins use translocons for their insertion and orientation in the membrane

• NEED TO CONTINUE

• Mechanism of transporting large molecules out of the cell; pathways detailed: ER to Golgi to PM.

• Protein synthesis and glycosylation occur predominantly in the ER Lumen (N-term)

Orientation: RER protein synthesis

• LDL Receptor

  • Uptakes LDL (Low density lipoprotein/lipid carrier in blood) = endocytosis

  • N-terminus: outside binds LDL receptor, facilitating the uptake of molecules

  • C-terminus: faces the cytoplasm, inside tiggers the uptake of signaling molecules

• Transferrin Receptor (Tf)

  • Uptakes Tf into the cell (iron carrier in blood) that binds to Fe3+ and transports it in circulation = endocytosis

  • Tf + Fe3+ internalized via clathrin-mediated endocytosis

  • N-terminus: inside tiggers

    • contains TM region that anchors the receptor in the PM

  • C-terminus: outside binds Tf

    • extracellular domain contains transferrin-binding site

  • acidic pH in endosomes: conformational change in C-term causes iron dissociation from transferrin

Protein Glycosylation (exocytosis)

protein stability, folding, and cell-cell recognition

intergral membrane proteins, lysosomal enzymes, and extracellular components

• when glycoprotein is folded correctly, the remaining glucose on its oligosaccharide chain is removed enzymatically and the glycoprotein is released from the chaperone

oligosaccharide synthesis/modification starts in Cytoplasm (C-Term)

• Plasma membrane TM proteins are synthesized in ER → golgi (secretion)

• 2 types

  • N-linked:

  • lipid carrier: dolichol phosphate (embedded in ER membrane)

    1. sugars added to dolichol phosphate by glycotransferases. (cystolic side) Attachment of N-acetylglucosamine followed by mannose residue

    2. flipped across ER membrane by transporter proteins

    3. assembled oligosaccharide is transferred to Asn

       • starts in cytoplasm

       • begins in the ER and continues to be modified in the golgi

       • enzyme: Glycosyl transferase

  • O-linked:

    • occurs after translation

    • oligosaccharide attached to Ser/Thr

    • occurs in golgi

      1. N-acetylgalacosamine is added to OH of serine or threonine residues. (doesnt require lipid carrier)

      2. sugars added by glycosyltransferases

Glycosylation and Quality Control

• Initiated in the ER, involving branched oligosaccharides and the role of chaperones in ensuring proper protein folding.

The Unfolded Protein Response (UPR)

• Mechanism to handle misfolded proteins in the ER, preventing cell death through specific pathways.

• if protein not folded properly → stop making/accepting proteins and make more chaperones to help with folding

• Working properly:

  • BiP chaperones bind to UPR sensors and keep inactive

• Lots unfolded proteins:

  • BiP released from UPR sensor = sensors activated → UPR

  • make active dimer

Quiz Question on ER Functions

• Identifies transcription as occurring outside of the ER, while translation of secreted proteins and glycosylation occur inside.

Secretory Pathway Overview

• Key proteins involved such as coat proteins (COPII, COPI, clathrin) and their roles in vesicle formation and cargo selection.

• motor proteins

  • move vesicles, interact w cytoskeleton,

• SNAREs and SNAPs

  • help vesicles fuse and dock

  -Coat Proteins

  • COP2

    • binds with ER membrane and help generate vesicles which moves from RER → golgi

    • anterograde vesicular movement

    • recruit coat proteins: small GTPase (Sar1)

      • coat protein is effector molecule that recruit cargo and promote curvature of ER membrane

      • vesicular structure with cargo molecules buds off

      • Sar1+GDP → inactive

      • GEF in ER membrane replaces/promotes binding GDP with GTP = active (Sar1+GTP)

  • COP1

    • tran-golgi → cis-golgi → ER

    • Retrograde movement

    • molecules that function in RER accidentally escape and need to be brought back to ER

    • KDEL (diff. a.a)

      • all outside because theyre charged

  • Clathrin

    • role in two pathways: Trans golgi → lysosome

    • facilitate endocytosis; moving cargo from extracellular to intra?

    • Triskelion structure: 3 heavy and 3 light chains

      • made of polypeptides and arrange in soccer-ball-like

    • Adaptor proteins:

      • mediate binding between clathrin and membrane receptors

• Importance of GTP versus GDP forms in the activity of small GTPases like Sar1.

The Golgi Complex

role in membrane trafficking

Cisternae: “stacks” that mature into different compartments (with different functions) where proteins and lipids are modified, sorted, and packaged for transport to their final destinations.

• cis-Golgi:

  • closer to nucleus/ER

  • receives proteins and lipids from ER

• trans-Golgi

  • furthest from nucleus/ER

  • sorts and packages vesicles to different destinations

• cis,medial,trans-Golgi:

  • protein processing

  • e.g. glycosylation and phosphorylation

• Cisternal Maturation model (Cisternae):

  • Retrograde movement of enzymes via vesicular transport (move back)

  • Cargo stay IN cisternae as they mature into different compartments

  • Evidence supporting model:

    • Immuno-gold EM techniques have provided visual confirmation of the presence of specific enzymes within the Golgi apparatus, demonstrating their retrograde transport back to the endoplasmic reticulum.

• Vesicular Model:

  • Enzymes stay put

  • vesicles carry the cargo through the cisternae

• Protein Processing

  • Proteolysis:

    • breakdown of proteins

    • e.g POMC

      • 1 gene, 1 transcript, 1 polypeptide,

  • Glycosylation

    • N-linked: modification in Golgi, involves attachment of oligosaccharides

    • O-linked: starts in Golgi

Types of vesicle transport in the Golgi Complex

1. COPII-Coated Vesicles:

   • ER → Golgi (Anterograde)

   • Coat assembly:

     • GEF in ER membrane: small GTPase (Sar1) binds to Sar1-GTP , which promotes the recruitment of COPII coat proteins.

       (GEF promotes small-GTPase to bind to GTP)

     • Coat proteins recruit cargo = membrane curvature

     • Sar1-GDP → INACTIVE

     • Sar1-GTP → ACTIVE

2. COPI-Coated Vesicles

   • trans-Golgi → cis-Golgi → ER (retrograde) between cisternae

   • Capture “escaped” ER proteins and return them to ER

     • ER proteins: KDEL receptors bind to COPI coat = retrograde transport

     • K (lysine) D (asparic acid) E (glutamic acid) L (leucine) all outside because charged

     • e.g. translocon would have KDEL sequence because it works in the RER

3. Clathrin-Coated Vesicles

   • transport proteins from trans-golgi to

     • Lysosomes (via mannose-6-phosphate)

     • endosomes

     • PM

Lysosomal Enzymes

• Trans-golgi → lysosome

  • sorting: other proteins secreted out and some go to lysozome (those are modified in golgi)

  • Mannose 6-phosphate

    • targets and modifies proteins, added in golgi

    • bind to enzymes and recruit them into vesicles

    • Clathrin coated vesicles:

      • GGA (adaptors): mediate between clathrin, MPRs, and lysosomal enzymes

      • Arf1 small GTPase bound to GTP = effector molecule AND active conformation

      • Arf1 effector: adaptor protein that recruits the clathrin coat proteins

• Recognition signals

  • phosphorylated mannose residues on N-linked carbohydrate chains

Transport through secretory pathway

• cytoskeleton organized

• motor proteins carry vesicles

Vesicle tethering and docking: w/ target membrane

• Tethers get vesicles close for docking (50-200 nm)

  • Rabs (small GTPase proteins) help recruit tethering proteins

    • help vesicles target specific compartments where they will be fused

• SNAREs: docking

  • v-SNAREs:

    • proteins on vesicle membrane

    • synaptobrevin protein

  • t-SNAREs

    • present on target membrane

    • SNAP-25

    • syntaxin

  • alpha-helical domains interact and pull vesicles to membrane

  • docked vesicles:

    • receive a signal → fuse with target (exocytosis)

  • Post-fusion

    • NSF (ATPase) disassembles SNARE complexes

Bacterial neurotoxins target SNAREs

• clostridium botulinum toxin

  • proteases (enzyme that breaks down proteins) break down SNAREs that prevent synaptic vesicles fusion = neurotransmitter cant be released = paralysis

Extra Notes

• membrane bound glycotransferases add sugar to dolichol phosphate

• biosynthesis pathway: ER → golgi complex → secretory vesicle → PM

• Organelle arrangement in secretory cell from basal → apical end

  • nucleus and RER → SER → golgi complex → secretory vesicles

• integral membrane proteins enter the lipid bilayer through the translocon channel gate that opens/closes continuously. Gives nascent polypeptide segment a chance to partition into hydrophobic core.

• incompletely/misfolded proteins display exposed hydrophobic residues that UGGT recognize

• misfolded secretory proteins destroyed in the cytosol