protein sorting

Stages from gene to functional protein

Transcription, translation, folding and transport

Why is energy needed during protein synthesis?

To regenerate aminoacyl-tRNAs and drive ribosome translocation

Is peptide bond formation energy-requiring?

No, it is spontaneous because energy is stored in charged amino acids

ATP cost per peptide added

2 ATP

GTP cost per peptide added

1 GTP

GTP

Guanosine triphosphate used in signalling, conformational changes and molecular switches, signal transduction mechanisms

ATP

Adenosine triphosphate used for conventional cellular energy costs

Why is protein degradation energetically favourable?

No energy is required to build the protein

Main challenge during protein folding

Hiding hydrophobic residues from water

Cause of protein aggregates

Exposed hydrophobic regions interacting with each other

Why do unfolded proteins aggregate?

Exposed hydrophobic surfaces interact and tangle together

Role of chaperones

Assist protein folding and prevent aggregation

How do chaperones prevent aggregation?

Bind exposed hydrophobic regions

Effect of chaperone binding to hydrophobic regions

Masks hydrophobic surfaces

Unfoldase activity

A chaperone activity that allows proteins to attempt folding again

Does chaperone unfoldase activity require energy?

Yes

What process of chaperone action is ATP-dependent?

Substrate release

Major family of molecular chaperones

Heat shock 70 family

Examples of Hsp70 proteins

Hsp70, Hsc70 and BiP

Location of Hsp70 chaperones

Cytosol and other cellular compartments

Protein-binding domain of Hsp70

Binds unfolded proteins

ATPase domain of Hsp70

Binds and hydrolyses ATP

Is protein folding energetically favourable?

Yes

Is protein unfolding energetically costly?

Yes

Passive function of chaperones

Prevent aggregate formation

Active function of chaperones

Dissolve aggregates and promote refolding

Quality-control role of chaperones

Target proteins for degradation and initiate unfolded protein response

Are chaperones part of the final protein structure?

No

Evidence for chaperone function

Mutant chaperones cause protein aggregation and failed refolding

Folding intermediate

A partially folded protein

Chaperone ligand during folding

A folding intermediate with exposed hydrophobic regions

Monomeric folding intermediate

Usually inactive

Dimerisation

Association of two protein subunits

Effect of dimerisation on chaperone binding

Protein is no longer recognised by chaperones

Why can completed folding still require chaperones?

Some proteins must assemble into complexes before being fully folded

Prokaryotic protein destinations

Cytosol or secretion across the plasma membrane

Relationship between transcription and translation in prokaryotes

Coupled

Why must secreted bacterial proteins unfold?

Folded proteins cannot pass through membrane pores

What places can a protein in a prokaryote end up?

Inner membrane

outer membrane

cytosol

Number of membranes in Gram-positive bacteria

One

Number of membranes in Gram-negative bacteria

Two

what is the protein targeting signal in bacteria known as?

Signal peptide

Function of bacterial signal peptides

Direct proteins for secretion

Fate of bacterial signal peptides

Cleaved after translocation

Example of prokaryote proteins for secretion?

Hydrolases; digest extracellular macromolecules

Relationship between transcription and translation in eukaryotes

Separate processes, not coupled

Fate of eukaryotic mRNA

Transported to cytosol for translation

Possible destinations of eukaryotic proteins

Mitochondria, chloroplasts, peroxisomes and secretory pathway

Port of entry into secretory pathway

Endoplasmic reticulum

Transport vesicle

Protein-coated vesicle moving cargo between compartments

Ribosome

Universal molecular machine that synthesises proteins from mRNA

Signal recognition particle (SRP)

Ribonucleoprotein that targets proteins to membranes

Function of SRP in eukaryotes

Targets proteins to the ER membrane, by recognising a signal peptide; via translocation

Function of SRP in prokaryotes

Targets proteins to the plasma membrane

Two possible fates of translated proteins

Secretory pathway or cytosolic localisation

SRP-dependent pathway

Protein enters secretory pathway

SRP-independent pathway

Protein remains in cytosol

Where does protein synthesis begin?

Cytosol

Exception to cytosolic protein synthesis

Chloroplast and mitochondrial proteins

Fate of proteins synthesised in cytosol

Fold and assemble after translation

Fate of proteins synthesised at rough ER

Fold, assemble and exit in vesicles

—> Exported to ER, translocation pore on ER sealed by ribosome

—> Depend on cytosolic mRNA

Co-translational translocation

Protein enters ER while still being translated

Advantage of co-translational translocation

Protein does not need to be unfolded for transport

Explain process of Co-transnational translocation

-The “signal recognition particle” (SRP) recognises “signal peptides” when they emerge as nascent chains
on the ribosome surface.
--> SRP stops or slows down translation by the ribosome

--> SRP targets the ribosome/mRNA/nascent polypeptide complex to the translocation pore on the ER

--> Engaged SRP's have an increased affinity to the ER Resident SRP receptor, which they bind and hand over ribosome-mRNA complex to translocation pore

--> Nascent protein inserts into aqueous channel of the pore and translation by the ribosome resumes

--> Protein fold up with ER lumenal  chaperones (BiP)

--> SRP and the SRP-receptor are being recycled back to cytosol (GTP hydrolysis),

 

-Cleavage of the signal peptide occurs well before the protein is fully translocated and is never part of the finally folded structure (The mature protein)

what happens after cotranslational translocation?

--> Ribosome seals a translocation pore on the rough ER

--> Protein needs to finish translation and folding within ER

-ER contains a special chaperone: The binding protein (BiP)

Why is BiP especially important in the ER?

High protein concentration increases aggregation risk

Role of BiP when pore is unused

Seals the translocation pore

Role of BiP during protein folding

Masks exposed hydrophobic regions

How long does BiP assist folding?

Until no hydrophobic regions remain exposed

Destination of correctly folded ER proteins

Golgi apparatus

Why do monomeric subunits bind BiP?

Exposed hydrophobic regions remain visible

When are multimeric proteins released from BiP?

After assembly to other proteins hides hydrophobic surfaces

Why do completed multimers escape chaperone binding?

Hydrophobic regions are buried in quaternary structure

Antibody subunits

Light chains and heavy chains

Where are antibody chains synthesised?

In the ER lumen

Why is antibody assembly into tetramers required?

To prevent continued BiP binding and allow secretion

Location of antibody signal peptides

N-terminus

What happens to antibody signal peptides?

They are cleaved after translocation

How do heavy chains interact with BiP?

Bind BiP until light chains assemble and form di-sulphide bridges

What stabilises assembled antibodies?

Disulphide bonds

Complex antibody example

IgM tetramers and pentamers

Destination of secreted antibodies

Bloodstream, soluble in the aqueous lumen of the endoplasmic reticulum

Membrane-anchored antibodies

Remain attached to membranes

Importance of membrane antibodies

Essential for immune system function

Location of antibody domain in membrane antibodies

Faces ER lumen, Golgi lumen, vesicle lumen or extracellular space

How is the light chain membrane-associated?

Through interaction with the heavy chain

Transmembrane domain (Protein insertion to membranes)

Hydrophobic stretch of amino acids

Are transmembrane domains cleaved?

No

Do transmembrane domains remain in mature proteins?

Yes

BiP discovery

Found bound to unassembled antibody heavy chains

GRP78

Protein discovered in glucose-starved cells,  where many proteins failed to fold properly due to missing sugar modifications.

Relationship between BiP and GRP78

They are the same protein

Function of BiP/GRP78

Assists protein folding and prevents accumulation of misfolded proteins

Importance of BiP discovery

Showed that cellular stress can reveal important proteins

Proteins destined for membrane insertion contain

A signal peptide and transmembrane domain

Explain process of protein membrane insertion

Final orientation of inserted membrane proteins

N-terminus in ER lumen and C-terminus in cytosol

Location of transmembrane domain

Embedded within the membrane

Why is ER lumen considered topologically outside the cell?

It lies beyond the final membrane barrier before secretion

Order of the secretory pathway

RER → Golgi → Vesicle → Secretion