M5L1 - Protein Processing and Trafficking RER

Dynamics of the ER

• Membranes of the ER constantly changes/undergoes

  • Shape/Structure

  • fisson/fusion

  • migration to new cell locations

RER (4) vs smooth ER (5)

Rough:

• Ribosomal complexes associate with it

• Site of co-translational transport

• Site of protein modification

• Formation of vesicles transporting proteins to GA

Smooth:

• No ribosomes

• Site of fatty acid synthesis

• Site of phospholipid synthesis

• Carbohydrate metabolism occurs here

• Calcium is sequestered/collected here to regulate [ca]

Post-Translational Modifications (PTMs) in ER (4)

• Occur along the length of entire protein if targeted to ER lumen 

• Occur along luminal portion if embedded in ER membrane 

Types:

• Glycosylation: Covalent addition of polysaccharides 

• Disulfide bond formation 

• Protein folding 

• Proteolytic cleavage 

Protein Glycosylation

• Addition of polysaccharide (sugar group) to protein 

• Common in

  • proteins to be secreted from the cell 

  • proteins embedded in cell membrane 

  • proteins which mediate cell interactions with extracellular matrix (space between outside of cells) 

  • proteins that mediate receptor-ligand recognition 

N-linked glycosylation in ER

• Most common form of glycosylation 

• Addition of a polysaccharide to the NH2 of asparagine’s R-group 

• This portion of the proteins remains on the luminal side 

• The modification appears on the exterior surface of the protein 

Disulphide bond formation in ER

• Covalent linkages between the (-SH) groups of two cysteine amino acids

• They provide added stability

  • Forms tertiary/quarternary structures of proteins

• Can occur 

  • Within single protein (intramolecular bond) 

  • Between proteins (intermolecular bond) 

• Common in proteins

  • secreted from the cell 

  • located on the outside surface of cell membrane 

• ER lumen has an oxidative environment favouring the formation of this bond (it’s an oxidation rxn) 

• Cytosol has a reducing env favouring the rev rxn 

RNAse A: Disulphide Bridge Protein Example

• has 4 disulphide bridges 

• Secreted into intestine to aid in digestion of RNA by cleaving it into smaller pieces 

• The bonds help maintain the structure in the acidic env of small intestine 

Protein Disulphide Isomerase (PDI)

• ER protein that promotes oxidation, thus promoting disulphide bridge formation 

• PDI can also correct inappropriate disulfide bridge formations

• Forms an intermediate with 2 cysteine residues to accelerate rate of rxn 

• Steps: 

  • Oxidized PDI contains a disulfide bridge 

  • PDI forms an intermediate with one cysteine residue 

  • This facilitates the formation of an intramolecular cysteine bond 

  • PDI is spontaneously converted back into the oxidized form bc of the oxidizing env of the ER luman 

Protein Folding in the ER (Lectins)

• Lectins assist protein folding by recognizing N-linked polysaccharides on them (from glycosylation) 

• They function similarily to molecular chaperones 

• Lectins types: 

  • Calnexin: Found throughout ER membrane 

  • Calreticulin 

BiP: HSP70

• Is an ER protein 

• The roles of BiP depend on its ability to recognize and bind to unfolded proteins 

• It binds to proteins as they appear in the ER through co-translational transport 

• BiP and it’s co-chaperones (Hsp40 and NEF) drive efficient ER protein folding 

• BiP initiates a process called the unfolded protein response in the ER 

Proteolytic Cleavage in the ER

• Cleavage of peptide backbone of protein

• Ex. N-terminal signal cleavage by signal peptidase 

• Ex. Proinsulin 

  • Has N-terminal sequence removed by signal peptidase 

  • Required to form the final insulin protein in pancreatic cells

  • Has disulfide bridges providing extra stability 

Proinsulin

• Has N-terminal sequence removed by signal peptidase 

• Required to form the final insulin protein in pancreatic cells

• Has disulfide bridges providing extra stability 

• Is the target of 3 other peptidases during transport in pancreatic cells through secretory vesicles 

Unfolded Protein Response (UPR)

• The RER can be flooded with unfolded proteins resulting from overproduction, delay in processing, exposure to toxins, denaturing, or lack of nutrients

• This causes a risk of aggregation and cell death

• The UPR is a response to this situation 

• First response: Try to restore normal function by slowing down new protein translation or removing unfolded proteins through ubiquitinylation 

• Second Step: Increase production of chaperone proteins assisting in folding 

BiP and Ire1 in UPR 

• BiP serves as chaperone to assist in folding and prevent aggregation 

• BiP is part of the system leading to the production of more protein-folding regulators 

• BiP and lre1 associated 

  • BiP is inhibited and lre1 is inactive

• Increase in unfolded proteins causes BiP and lre1 to disassociate

  • BiP has higher affinity for hydrophobic pathes on unfolded proteins

• lre1 forms homodimers in the ER which serve as activated endonucleases 

• Endonucleases make internal cuts in nucleic acids like mRNAs

Hac1 Expression during UPR 

• lre1 endonuclease targets the mRNA for Hac1

• Unspliced Hac1 mRNA has an internal sequence inhibiting translation by ribosome 

• lre1 splices Hac1 mRNA to allow for the synthesis of Hac1 protein 

• It serves as a transcription factor 

  • It’s transported to the nucleus to activate the transcription of several genes

  • This includes BiP, lectins, PDI and signal peptidases 

Protein Trafficking Away / To ER

• Anterograde Transport: Proteins move from ER to the Cell membrane (away from ER) 

• Retrograde Transport: Proteins brought back to ER 

Techniques for Studying Vesicular Transport (from ER to out of cell)

• Pulse-chase labeling and visualization using immuno-TEM

• Fluorescent microscopy of GFP-labelled proteins 

  • Both in mammalian cells 

• Genetiv mutations disrupting transport in yeast cells allowed for identification of the proteins required 

Pulse-Chase Labeling: Acinar cells

• Acinar cells: exocrine cells of the pancreas

• they produce and transport enxymes secreted into digestive system 

• Tagging the secreted enzymes can show us where they go after leaving the ER 

Pulse-chase:

• Tagging proteins for a brief amount of time so only some are labelled 

• Acinar cells were incubated in a medium with radioactive methionine 

• This is a ‘pulse’ of labelling which lasted 3 mins 

• Some cells are removed from the medium, washed, and transferred into a medium with non-radioactive amino acids

• This ‘chase’ can vary in length so proteins can be visualized at different stages in their journey from RER to secretion 

Pulse-Chase Labeling Different Chase Lengths

Time Point 1:

• Cells grown in radioactive medium for 3 mins

• They’re fixed immediately with no ‘chase’

• They’re visualized using electron microscopy

• They’re found in the ER bc they haven’t moved

Time Point 2: 

• Cells grown in radioactive medium for 3 mins

• Allowed to be in unlabelled medium for 17 mins (protein transport occurs) 

• They’re then fixed and visualized 

• They’re found in the GA

Time Point 3: 

• Cells grown in radioactive medium for 3 mins

• Allowed to be in unlabeled medium for 117 mins

• They’re then fixed and visualized

• The proteins are found in secretory vesicles

Pulse-Chase Graph: Location of Labeled Proteins Throughout Experiment 

• y axis: % of autoradiographic grains on TEM image (due to radioactively-labelled proteins)

• x axis: time (length of chase) from 0-120 mins 

GFP Tagging of VSV (Vesicular Stomatitis Virus)

• Carries a gene coding for an envelope protein (G protein) 

  • Viral proteins can be synthesized and embedded in host membrane 

  • It becomes part of the viral envelope surrounding the virus then 

• A mutant VSV-G protein can be tagged 

• At permissive temp (32C) the protein variant can fold and will be found in cell membrane 

• At restrictive temp (40C) the protein variant denatures and is retained in ER by UPR quality control 

Pulse-Chase GFP Tagging of VSV (Vesicular Stomatitis Virus)

• Infect mammalian cells with viruses carrying VSV-G:GFP gene with enough time to induce protein synthesis 

• Disable the virus and culture the mammalian cells at 40C

  • VSV-G:GFP is retained in ER 

• Lower temp to 32C

  • VSV-G:GFP will fold and be transported out 

Tracked movement

• 0 mins: temp not shifted for protein accumulates in ER

• 40 mins after move to 32C: Fluorescent protein moved to GA

• 180 mins after move to 32C: fluorescent protein moved to membrane

Graph:

• Summarizes location of VSV-G:GFP protein 

• y-axis: amount of fluorescence in cell in arbitrary units 

• x-axis: length of chase from 0-600 mins 

• Fluorescence dec overtime as fluorophores lose their fluoresence as they move from ER to GA to membrane

Saccharomyces cerevisiae (yeast) model in determining steps in protein transport 

• It was for protein transport to cell membrane from ER 

• The yeast S. cerevisiae metabolizes sucrose by hydrolyzing it into glucose and fructose 

• This is done by protein invertase created by another cell 

• Defects in protein transport pathways can be identified by following the secretion of invertase

Invertase Secretion:

• Generated random mutations in yeast genome

• They looked for temp-sensitive mutations than failed to secrete invertase in restrictive temps 

  • The mutations code for proteins folding normally at permissive temp but not in restrictive temp 

• A restrictive temperature shift will reveal defect in transport (invertase will accumulate in vesicles) 

• Each gene mutation was called a sec mutant (secretory mutant) 

• They were named by number given the place of mutation 

  • Ex. Sec61 (translocon protein) 

Class A sec mutants (yeast experiment)

• Invertase accumulated in different locations based on which step was defective

• Different classes were identified based on the location of accumulation 

• Class A showed accumulation in cytosol

  • Normally it would be in the ER, GA, vasicles, and outside the cell 

• It suggested the mutation prevents the first step of co-translational transport

• This would be linked to any component of ER translocon 

  • SRP protein 

  • SRP receptor 

  • signal sequence 

  • Sec61-alpha (ER translocon)

The 5 Classes of sec mutants (yeast invertase study)

• Class A: Accumulates in cytosol

• Class B: Accumulates in ER

  • Defect in vesicle transport out of ER

• Class C: Accumulates in ER to Golgi transport vesicles

• Class D: Accumulates in in GA

  • Defect in forming vesicles to leave GA

• Class E: Accumulates in secretory vesicles to secrete out of cell

Double Mutants in Yeast Experiment to Determine Protein Transport 

• Cells carrying 2 mutations from a different class each 

• This could demonstrate the step-wise progression of transport 

• Ex. Cell with Class A and B mutations would only show a Class A phenotype 

  • Therefore, Class A proteins are needed before Class B ones

• Upstream mutations mask the appearance of downstream phenotypes

• A collection of double-mutants confirmed the pathway from RER to GA to vesicles