Week 7 - Moving Proteins / Translation Localization

What classic experimental tools were used to discover the process of ER protein translocation? - 4 main points

• Microsomes:

  • A paper proved that amylase protein only existed inside microsomes therefore there must be some mechanisms for getting the proteins inside the cell

  • Another paper found that if you insert an RNA in a solution with microsomes, if the protein is destined for the secretory system it will be co-translated inside the microsome

• Co-Translation / translocation

  • 1970’s experiment showed proteins getting clipped

  • IF you translate the mRNA with ribosomes THEN add microsomes, no protein will be inside the lumen and product is larger

  • IF you add microsomes THEN mRNA, proteins are co-translated inside the lumen and product is smaller 

  • Results indicate that in order for secretory proteins to get into the ER, it must happen during translation and the process clips a small part of the protein off (signal sequence)

• Purifying translocon

  • purified with high salt concentration and elastase

    • High salt concentration allows separation of complexes held together by electrostatic interactions just a little bit

      • NOTE: too much salt causes the complexes to stick together 

    • Elastase is a protease that digests peripherally associated proteins from the membrane without damage

• Detergents:

  • Sodium deoxycholate: gentle detergent that doesn’t denture proteins

  • Used to solubilize microsomal membranes without damaging luminal cargo - allow for the investigators to release and analyze proteins that have been translocated into the microsome

• All of these were used to map and characterize the translocation pathway.

Microsome

Mini rough ER that can translate mRNA into secreted proteins

Given a secreted protein, which pool of ribosomes translates it and why?

Ribosomes bound to the rough ER translate secreted proteins because the nascent peptide carries an N-terminal ER signal sequence that recruits SRP and moves the ribosome to the ER membrane.

List the correct order of molecular events from transcription to a folded ER luminal protein.

DNA transcription → pre-mRNA processing (capping, splicing, poly-A) → export to cytosol → translation begins → N-terminal ER signal engages SRP → ribosome docks to ER via SRP receptor & Sec61 → co-translational translocation into ER → signal peptide cleavage → folding with ER chaperones & enzymes → final ER-resident folding or membrane insertion.

(LO: sequence gene expression → folded ER/tm protein)

Name / describe the ER —> 

• targeting sequence

• Receptor

• Translocation channel

• Energy source  

• Targeting sequence: 6-12 a.a., preceded by one or more basic a.a. like Arg and Lys

• Receptor: SRP recognizes target sequence and brings complex to SRP receptor —> both SRP and SRP receptors are GTPases 

• Translocation channel: Sec61 

• Energy source: GTP hydrolysis that powers elongation of translation 

Name / describe the Mitochondria —> 

• Receptor:

• Translocation channel:

• Energy source:

• Receptor: Tom20/22

• Translocation channel:

  • Tom40: transport in the outer mitochondrial membrane 

  • Tim23: transport in the inner mitochondrial membrane

• Energy source: ATP hydrolysis by Hsp70 in the matrix (also helps keep protein unfolded during this process)  

Name / describe the Peroxisome —> 

• Receptor:

• Translocation channel:

• Energy source:

• Receptor: Pex 5

• Translocation channel: Pex14

• Energy source: ATP hydrolysis coupled to ubiquination and deubiquination of Pex5

Name / describe the Nucleus —> 

• Receptor:

• Translocation channel:

• Energy source:

• Receptor: nuclear transport receptors

• Translocation channel: Nuclear poor which is filled with a diversity of different proteins that monitor what goes in and out

• Energy source: GTP hydrolysis coupled to cycling Ran GTPase into and out of the nucleus

What is the only strictly conserved rule about ER signal sequences?

For ER targeting, the signal sequence must be located on the N-terminus

• Other sometimes/mostly true - 6-12 (sometimes 22) a.a. that are followed by basic amino acids

(LO: ER targeting — general nature of signal sequence)

Compare the receptor for ER targeting vs mitochondrial targeting.

ER uses SRP + SRP receptor

Mitochondria use surface receptors such as Tom20.

Why do luminal, secreted, and TM proteins have ~6–12 fewer amino acids than their translation products?

Their signal peptide is cleaved off by signal peptidase during ER insertion.

Contrast eukaryotic vs bacterial SRP & composition

Eukaryotic SRP:

• Structure: 6 different proteins + 300 nu 

• SRP: soluble + GTPase

• SRP Receptor: membrane bound + GTPase

• Translocon: Sec61

Prokaryotic SRP:

• Structure: 1 Protein Ffh* + 114 nu

• SRP: Soluble + GTPase

• SRP Receptor: Soluble + GTPase - FtsY

• Translocon name: SecYEG

(LO: compare eukaryotic vs bacterial SRP — function)

What prevents ATP and Ca2+ leakage at the ER and how?

Sec61

Contains a plug domain and has hydrophobic isoleucine “gasket” that restricts non-polypeptide passage

What is th ER Folding Timer

• Proteins co-translated into the ER lumen have a oligosaccharide attached to asparagine residue

  • Oligosaccharides contain 2 N-acetylglucosamines, mannoses, and 3 glucoses

• The number of glucoses attached acts as the timer for protein folding

• Glucosidase I and II pick of the glucose

• When one glucose is left chaperons bind to protein and detects misfolded / unfolded proteins

(LO: describe ER folding timer — oligosaccharide addition)

What is the fate of misfolded proteins upon the last glucose being trimmed 

• Protein is not finished folding and UGGT will add more glucose to extend timer

• Protein is not finished folding and ER alpha mannosidases (trims mannose) degrade protein

What experimental result demonstrated that ER translocation is co-translational and not post-translational?

Secreted proteins are only found inside microsomes when microsomes are present during translation; when added afterward, proteins remain cytosolic and are larger because the signal peptide was never cleaved.
(LO: discovery of ER translocation mechanism)

How was signal peptide cleavage inferred before signal peptidase was known?

Proteins translated in the presence of microsomes migrated with a lower molecular weight on SDS-PAGE than those translated without microsomes, implying clipping of an N-terminal segment.
(LO: discovery of ER translocation mechanism)

Given a mitochondrial matrix protein, which pool of ribosomes translated it?

Free ribosomes in the cytosol — nuclear-encoded mitochondrial proteins are not translated at the ER.
(LO: identify ER vs cytosolic ribosome pools from destination

Given a secreted hormone, which pool of ribosomes translated it?

ER-bound ribosomes, triggered by an N-terminal signal peptide recognized by SRP.
(LO: identify ER vs cytosolic ribosome pools from destination)

Is mitochondrial targeting also always at the N-terminus?

Often, but not universally — some organelle signals can be internal; only ER targeting strictly requires N-terminal positioning.
(LO: compare signal-based targeting principles)

What evolutionary inference is supported by the structural similarity between bacterial and eukaryotic SRP?

The conservation suggests eukaryotic SRP evolved from a bacterial ancestor, indicating the mechanism is ancient and essential.
(LO: compare eukaryotic vs bacterial SRP — evolutionary conservation)

What is the key functional similarity between bacterial and eukaryotic SRP systems?

Both SRP systems recognize signal peptides, pause translation, and deliver ribosome–nascent-chain complexes to membrane translocons for insertion.

Both: contain hydrophobic groove that binds to target signal sequence
(LO: compare eukaryotic vs bacterial SRP — function)

Describe the Type I transmembrane protein

• N-terminus faces exoplasmic/lumenal side; C-terminus faces cytosol.

• Normal co-translation as soluble protein until translocon recognizes a stop-transfer anchor sequence

• Conformational change causes ribosome to disassociate with translocon and continue translating protein in the cytosol 
  (LO: protein targeting to ER — transmembrane protein mechanisms)

Describe the Type II transmembrane protein

• N-terminus faces cytosol; C-terminus faces lumen/exoplasmic side.

• Signal sequence is in the middle of the protein and not directly on the N-terminus

• Ribosome translates mRNA as usual until the signal sequence pops out where it is then brought to translocon and the rest of the protein loops into the ER

• (LO: protein targeting to ER — TM insertion logic)

How does a signal-anchor in Type II proteins differ from the cleavable ER signal of soluble proteins?

Type II signal-anchors are internal and not cleaved; they themselves become the membrane-spanning segment.
(LO: differentiate stop-transfer vs signal-anchor)

Stop Transfer Anchor Sequence

14 - 15 hydrophobic residue that halts translocation and anchors the segment in the bilayer

(LO: protein targeting to ER — stop-transfer sequences)

Why must mitochondrial-destined proteins remain unfolded prior to import?

Cytosolic Hsp70 uses ATP to prevent premature folding so the polypeptide can thread through TOM/TIM pores.
(LO: sequence events — mitochondrial targeting mechanism)

How does nuclear import/export uniquely differ from ER/mitochondrial/peroxisomal import with respect to folding state?

Proteins imported/exported through nuclear pores are fully folded; other systems typically require unfolded translocation.
(LO: compare organelle targeting — mechanistic distinctions)

How do Ran-GTP and Ran-GDP differentially regulate importins?

Nuclear Transport

Ran-GTP binds importin in the nucleus causing cargo release; Ran-GDP predominates in cytosol allowing importin to bind new cargo.
(LO: compare Ran-GTP/GDP effects — importin vs exportin)

How do Ran-GTP and Ran-GDP differentially regulate exportins?

Ran-GTP promotes cargo binding to exportin in the nucleus; hydrolysis to Ran-GDP in the cytosol causes cargo release.
(LO: compare Ran-GTP/GDP effects — importin vs exportin)

What do glucosidases I and II do in ER quality control?

They trim glucose residues from the oligosaccharide, controlling the time window for chaperone binding.
(LO: roles of glucosidases in folding window)

What is the role of calnexin and calreticulin in ER protein folding?

They bind partially trimmed glycoproteins (lectin chaperones) to assist proper folding before release.
(LO: roles of calnexin/calreticulin in timer)

What is the role of UGGT in the ER folding cycle?

UGGT re-adds glucose to incompletely folded proteins, re-entering them into another calnexin/calreticulin cycle.
(LO: time-extension mechanism via UGGT)

What molecular signature targets a protein for ERAD?

Progressive mannose trimming after failed folding cycles commits the protein to ER-associated degradation.
(LO: ERAD outcome in folding timer)

What is BiP’s role in the ER?

BiP (Hsp70 family) binds hydrophobic patches to keep chains unfolded or prevent misfolded aggregation during folding.
(LO: compare chaperones — BiP)

What is the role of protein disulfide isomerase (PDI)?

PDI reshuffles incorrect disulfide bonds, ensuring the protein reaches the correct native disulfide pattern.
(LO: compare PDI vs chaperones)

What is the function of oligosaccharyl transferase (OST)?

OST transfers the pre-assembled oligosaccharide from lipid precursor to nascent polypeptides entering the ER.
(LO: compare OST vs chaperone vs PDI roles)

How are GPI-anchored proteins produced from Type I membrane precursors?

A Type I TM protein is cleaved luminally by GPI-transamidase; the new C-terminus is covalently linked to a pre-formed GPI anchor.
(LO: describe GPI-anchor formation)

How do BiP, calnexin, calreticulin, and Hsp70 differ in function from protein disulfide isomerase and oligosaccharyl transferase?

BiP, calnexin, calreticulin, and Hsp70 act as chaperones that bind nascent or misfolded proteins to prevent aggregation and assist correct folding;

PDI catalyzes disulfide reshuffling to correct bond pairing;

OST transfers a pre-built oligosaccharide onto nascent chains as the timing scaffold for ER quality control.