MCB 2210 L14: Protein Targeting: Endoplasmic Reticulum Part 1

Slides 1-33

Describe where and how protein synthesis primary occurs + main pathway

1. Protein synthesis @ cytosolic/free ribosomes

   1. Has targeting sequence (address)?

      1. NO → stays in cytosol

      2. YES → guided to specific destination (Mitochondrion, nucleus, chloroplast, endoplasmic reticulum)

2. Main pathway = ER/Secretion

   1. HAS signal peptide (address)

   2. Co-translational process

      1. Ribosome → ER WHILE protein synthesis occurs

         1. ER = moving bilayer

      2. Protein pushed through pore (translocator) into ER lumen

         1. ER = moving proteins

   3. Protein fold inside ER → vesicles → Golgi apparatus

      1. Golgi sorts protein to final destination (Plasma membrane/lysosome)

Where are lipids and proteins synthesized in the ER sent? What is this called?

Lipids & proteins synthesized in ER → secreted to plasma membrane/outside world

Constitutive secretion: continuous, unregulated, & default pathway present in all eukaryotic cells that transports proteins & lipids from Golgi apparatus directly to plasma membrane for release

How are proteins targeted to the ER?

1. Signal sequences @ beginning of protein translation

2. Signal Recognition Particle (SRP) binds to sequence → PAUSES translation → ribosome directed to ER membrane

3. SRP binds to SRP receptor on ER membrane → ribosome → translocon

   1. Translocon = protein channel in ER membrane = gateway for newly made proteins

4. SRP releases ribosome + protein complex → translation resumes → chain pushed through translocon channel into ER lumen or membrane

5. Signal sequence clipped by signal peptidase → finished protein released into ER by ribosome

What are the 3 ways proteins are moved between compartments within a eukaryotic cell?

1. Gated Transport = Cytosol → Nucleus (Nuclear Pore Complexes)

   1. Traffic = bidirectional

2. Transmembrane Transport = Cytosol → non-nuclear organelles (Mitochondria, Peroxisomes, Plastids, ER)

   1. Proteins = unfolded → threaded through protein translocated in organelle’s membrane

3. Vesicular Transport = Secretory Pathway

   1. Protein enters ER → carried through vesicles from:

      1. ER → Golgi → Late Endosome/Lysosome or Secretory Vesicles → Cell Surface

   2. Protein never re-enters cytosol (protein stays inside vesicle)

Which cellular compartments are considered “topologically equivalent” to the extracellular space, and why does this matter for vesicular transport?

• Topology equivalent compartments = compartments where molecules can move b/w them w/o having to cross a physical membrane

• “Equivalent” Compartments = nuclear envelope, lumens of ER, Golgi apparatus, endosomes, lysosomes, and peroxisomes

  • Form endomembrane system = collection of interrelated membranes & organelles that work together to synthesize, modify, package, and transport proteins & lipids

• Vesicular transport = allows proteins to move between “topologically equivalent” compartments

• Secretory vesicle fuses w/ plasma membrane → vesicle’s interior = cell’s exterior

What are the 2 functions of the Endoplasmic Reticulum (ER)?

1. Synthesis for endomembrane system

   1. Proteins in plasma membrane, transmembrane, GPI-linked (lipid-anchored), soluble proteins in lumen of endomembrane system

2. Storage

   1. Calcium (Ca²⁺)

   2. Enzymes (some cell types)

      1. Ex. detoxifying enzymes in liver cells

ER as a dynamic structure

ER = moving network of tubules & sheets

• Stretches from nucleus to outer periphery of cell (plasma membrane)

• During cell division:

  • ER breaks into → vesicles → split into daughter cells

Endoplasmic reticulum structure + location within cell

Two types of ER membranes (viewed through electron microscope)

1. Rough ER = studded with ribosomes → rough → produces & process proteins

   1. Closer to nucleus

2. Smooth ER = NO ribosomes → smooth → produces lipids, steroids, & detoxifies drugs

   1. Peripheral (outer) regions of cell

How are ribosomes bound to the Rough Endoplasmic Reticulum membrane?

• Ribosomes bound through nascent chains

  • Nascent chains = protein molecule in process of being synthesized by ribosome

  • Proteins = co-translationally inserted into membrane

1. Signal Recognition Particle (SRP) recognizes specific signal sequence on protein → pauses translation

2. Ribosome + nascent protein chain directed to SRP receptor on ER

3. Ribosome = anchored to ER membrane by translocon

   1. Translocon = protein channel in ER membrane

4. Translation resumes when ribosome = bound to translocon

   1. Pushes protein directly into lumen → embedded in ER membrane

Describe how a cell sample is prepared for gradient centrifugation and the two types of ER membranes that can be separated

1. Mechanically break cell → microsome formation

   1. Microsomes = small, artificial vesicles created when cells are broken in lab (formed from ER fragments)

Rough Microsomes = DENSE due to ribosomes

Smooth Microsomes = less dense due to lack of ribosomes

• Centrifuged in sucrose density gradient

Microsome

Fragments of rough or smooth ER that seal themselves into tiny, artificial vesicles when cells are mechanically broken in a lab.

Free vs. Bound Polyribosomes

Protein translation = slow process

mRNA = long

Multiple ribosomes can attach to a single mRNA = polyribosome/polysome

Polysome = single mRNA molecule being read by multiple ribosomes simultaneously. Appears like “beads on a string”

• Protein HAS ER targeting signal ribosome & growing peptide chain (nascent chain) go to ER membrane → bound polyribosome/polysome

• Difference b/w free vs. bound polyribosomes = localization & peptide synthesized

• Rough Endoplasmic Reticulum (RER) = efficient

  • mRNA near membrane (many ribosomes = recycled)

ER signal sequence

ER signal sequence = hydrophobic amino acids

• N-terminal signal sequence

• Sometimes label = internal for membrane proteins (hydrophobic)

• Necessary & sufficient for ER membrane targeting

  • Only need sequence → ER membrane

• Co-translational translocation across ER membrane = protein transported to ER as protein = translated

• Signal peptidase = cleaves ER signal sequence after targeting

  • Signal ≠ found in mature protein

• Protein = folds in lumen w/ chaperones + other molecules

• Protein = free → soluble in ER lumen OR bound to ER membrane

What is ER targeting signal recognized by?

Signal Recognition Particle (SRP): factor that allows attachment of pure ribosomes to ER membranes

• Ribonucleoprotein particle = RNA + 6 proteins

• Binds to signal sequence & ribosome

• Binding of SRP to nascent peptide chain → stops translation (peptide = 70-100 AA long) = long enough to protrude from ribosomes

Co-translational translocation of proteins → ER

• Signal sequence = out of ribosome

  • Associates w/ Signal Recognition Particle (SRP)

    • Binds to signal sequence + ribosome

• SRP associates with SRP receptor in membrane

  • Ribosome docks onto translocon channel

    • Protects proteins from protease in assays

• Translocon pore though membrane opens (stays closed to prevent leakage of small molecules)

  • Signal sequence + nascent chain → pore

  • SRP + SRP receptor RELEASED from ribosome & translocon

  • Translation proceeds through membrane pore → protein enters lumen

• GTP binding/hydrolysis of BOTH SRP + SRP receptor regulate:

  • SRP/SRP-receptor binding

  • Ribosome docking

  • SRP/SRP-receptor release

  • Process is not well known

• Signal peptidase cleaves N-terminal ER signal sequence

• Protein synthesis = completed (ribosome docked)

• Translocation channel closes → ribosome undocks

How do you demonstrate co-translational transport?

Use microsomes & protease protection experiments → determine if proteins = exposed

• Mix mRNA encoding ER-targeted protein + ribosomes + ATP + tRNAs + Amino Acids = synthesize protein (in vitro or in lab)

• During/after protein synthesis + protease → protein = degraded by protease (through gel)

• During/after protein synthesis + protease + Microsomes → protein ≠ degraded

• During/after protein synthesis + detergents → protein = degraded

Conclusion:

• Proteins = protected during synthesis

  • Proteins = transported to lumen of membranes DURING synthesis → co-translational translocation

Explain what is happening in this picture

• Lane 1 = Protein synthesized → microsome

  • Protein = safe from protease inside microsome

• Lane 2 = protein synthesized while protease = present

  • Ribosome plugged into microsome → protein threads into microsome

  • Protein = safe from protease inside microsome

• Lane 3 = detergent added → microsome membrane = dissolved

  • Protein = exposed ≠ safe from protease

Lanes 1-3 = smaller than Lane 4 (farther along lane) → N-terminus signal sequence = CLEAVED OFF by signal peptidase

• Lane 4 = protein synthesized w/o microsomes

  • No microsomes = no signal peptidase → N-terminal signal sequence = STILL ATTACHED

  • Only appears if no protease is added

• Lane 5 = + protease

  • No microsomes = protein = unprotected ≠ protected from protease

How is the full sequence of events of protein targeting to ER demonstrated?

By using purified components

• A = (-) SRP, (-) SRP receptor, (-) microsomes

  • = Complete polypeptide w/ signal sequence

• B = (+) SRP, (-) SRP receptor, (-) microsomes

  • = Polypeptide elongation paused @ 70-100 AA

• C = (+) SRP, (+) SRP receptor, (-) microsomes

  • = Complete polypeptide + signal sequence + SRP & SRP receptor released from polypeptide (GTP hydrolysis)

• D = (+) SRP, (+) SRP receptor, (+) microsomes

  • = Polypeptide synthesized in microsome through transmembrane channel + signal sequence = cleaved + found in microsomes

Purified components + protease protection

Protein Outcomes:

• Ribosome → (+) full length protein, (-) protease protection

  • Protein = floats in liquid

• Ribosome + SRP → (-) full length protein, (+) protease protection

  • SRP = pauses translation @ 70-100 amino acids

• Ribosome + SRP + Receptor → (+) full length protein, (-) protease protection

  • SRP = released from protein

  • No membrane of microsome present to go inside

• Ribosome + SRP + Microsome → (+) full length protein, (+) protein protection

  • Protein threaded into → microsome

  • Shorter because signal sequence = cut off (signal peptidase)

Stripped Microsomes (sMicro) = microsomes (-) ribosomes (-) SRP (+) SRP receptor (+) translocon (+) signal peptidase

• SRP = chemically removed → Protein = outside (sensitive to protease)

Rough Microsomes = contain “hitchhiking” SRP

• Protein can go inside (protected from protease)

SRP is always required for translocation

Signal peptidase

Protease that recognizes N-terminus signal sequence

• Embedded in ER membrane & associated w/ translocon complex

Releases soluble proteins into lumen of ER

• Can be secreted or reside in ER, Golgi, lysosomes (w/ appropriate signals)

• Signal ≠ removed → protein stuck in membrane held by signal sequence