BIOL 201 - 9. Protein Targeting to the ER and Secretion

Proteins made in the ER

• Simultaneous protein synthesis and translocation across ER membrane (co-translational import)

• Protein folding and modification in the ER

• Protein transport to final destination via vesicles

Protein Synthesis Location

Cytoplasmic free ribosomes

Endomembrane System Proteins can be found in

ER, Golgi, Vacuole, Lysosome, Plasma Membrane

• soluble or membrane bound

Intracellular Transport

Transport within the cell and movement across membranes, with protein cargo using signal sequences and all processes requiring energy

1. through nuclear pores

2. using membrane translocators

3. using transport vesicles

Secretory System Proteins Process

N-terminus of newly synthesized protein acts as an ER signal sequence

• first 16—30 amino acids

• bind to molecules that then translocate the protein to the ER

Protein Targeting Process

1. N-terminal signal sequence on new protein emerges from the ribosome

2. Signal sequence binds to signal recognition particle (SRP)

3. SRP transports ribosome, mRNA, protein complex to SRP receptor on the ER membrane

4. Binding of SRP to its receptor is enhanced when both are bound to GTP

5. SRP and SRP receptor hydrolyze GTP to GDP

6. Signal peptidase cleaves N-terminal signal sequence

7. Ribosome remains attached to translocon

8. Translation is completed

Glycolysation

The covalent addition and processing of carbohydrates

Synthesis of Membrane Proteins

Integral and membrane associated proteins found in the ER, Golgi, Lysosome, PM, are inserted into the membrane during protein translation

• sequences within the proteins direct correct membrane insertion and orientation

• ER signal sequence is still required

• hydrophobic amino acid sequences become the membrane spanning domains of the protein

Protein Modifications in the ER

• Glycolysation

• Formation of disulfide bonds

• Folding of polypeptide chains and assembly of multisubunit proteins

• Specific proteolytic cleavages (ER, golgi, secretory vesicles)

Proteins created on the rough ER

Core Glycosylation

Glycosylation in which one or more carbohydrate chains are added to specific amino acids within all ER proteins

N-linkages

A carbohydrate chain that adds a complex branching sugar structure

• most common linkage

• sugars attached to amide nitrogen in asparagine

O-linkages

Sugars attached to hydroxyl groups in serine and threonine amino acids

• complex consists of 1-4 sugar residues

Glycosyltransferases

Enzymes that create sugar chains and add them to target proteins

• removed by glycosidases

• golgi resident protein

Glycosylation of Proteins in the ER

The sugar branch is transferred from dolichol phosphate to sites within newly synthesized proteins. The sugar addition facilitates appropriate folding, protein stability, recognition, binding, and adhesion

• glycosidases remove specific sugars

• further sugar modifications occur in the ER and golgi

BiP

A molecular chaperone protein that binds to proteins as they enter the ER

• prevents segments of the intermediate protein chain from misfolding or forming aggregates

PDI

An enzyme that forms disulfide bonds and confers stability and proper folding conformations

• protein disulfide isomerase

Calnexin and Calreticulin

Lectins that aid in folding

Degradation of Misfolded ER Proteins

Misfolded proteins leave the ER and undergo an unfolding protein response

• ubiquitinated and targeted for degradation in the proteosome

Secretory Pathway

The travel route for soluble and membrane proteins that reside in the PM, lysosome, golgi, and ER, or that are secreted from the cell

Endocytic Pathway

The route used to take up substances from the cell surface to the interior of the cell

Common Mechanism between Secretory and Endocytic Pathways

The transport of both membrane and soluble proteins occurs via transport vesicles between membrane bound compartments

Vesicles

Small, membrane bound sacs used to transport proteins between organelles

• buds from membrane of “parent” or “donor” organelle

• “dock” to target

• fuse with target or recipient organelle

• connects cellular compartments during secretion

• travels along cytoskeletal pathways

Coat Proteins

Molecular complexes that drive intracellular membrane transport and cargo selection by assembling on membranes to form vesicles

• controlled by a GTP-binding protein, that when switches to GTP, initiates coat polymerization

• COPII - controlled by Sar1-GTP

• COPI and Clathrin - controlled by ARF-GTP

Clathrin

A coat protein formed by ARF and pinched off by Dynamin

• cargo interacts with AP and clathrin coat proteins

• vesicles pinch off after budding

Targets the vesicle from the golgi to endosome, and plasma membrane to endosome

SNAREs

Proteins involved in recognition and fusion that form coils/coil complexes that “tie” two membranes together

• v-SNARE (vesicle) is uncovered

• can interact with appropriate t-snare (target) on target membrane

Rab Proteins

G-proteins that help in targeting vesicles to the appropriate membrane and interacts with effectors that are attached to target membranes to dock and tether to the target

• when switch to GTP form, Rab can insert its lipid anchor into the vesicle membrane

• SNAREs interact

• the vesicle is already uncoated, v-SNARE exposed

COPII

A coat protein that targets the vesicle form the ER to the golgi

• controlled by Sar1-GTP

COPI

A coat protein that targets the vesicle from the golgi to the ER and golgi to golgi (retrograde transport)

• controlled by ARF-GTP

Motor Proteins

Proteins that move along cytoskeletal pathways to transport vesicles

• changes shape using ATP hydrolysis to move

• Dynein: plus to minus (Golgi to ER, endosomes, PM)

• Kinesin: minus to plus (ER, PM, Lysosome to Golgi)

Anterograde Transport

Intracellular transport that moves cargo from the ER to the golgi, lysosome, PM, and for secretion

• COPII

• cytoplasmic targeting sequence: DXE

Retrograde Transport

Intracellular transport that moves cargo from the golgi to the ER, retrieving v-SNARE proteins, missorted ER resident proteins, and the membrane itself to make more vesicles

• COPI

KDEL Receptor

A C-terminal signal peptide that returns missorted ER resident proteins from the golgi back to the ER

• binding is regulated by pH, binds to ligand at low pH in the cis-golgi

Glycosidases

Golgi resident proteins that remove sugar chains from proteins

Lysosomes

Organelles for the degradation of components of a cell or organism

• late endosomes and phagosomes fuse with lysosomes

Early endosome → Late endosome → Lysosome

M6P (Mannose 6 Phosphate)

A targeting sequence for soluble lysosomal enzymes. At TGN, M6P receptors bind M6P residues on lysosome destined proteins

• generated by N-linked oligosaccharides undergoing a 2 step reaction in cis-golgi

• ligand binding to M6P receptors is enhanced at pH 6.5, reduced at pH of 5

• phosphatase in late endosomes removes phosphate from M6P residues (prevents rebinding to M6P receptor)

Receptor Mediated Endocytosis

Endocytosis where ligands bind to specific receptors and complexes are incorporated into clathrin coated vesicles and internalized