8. Protein Synthesis, Modification and Trafficking

Protein Synthesis, Modification and Trafficking – How do They Know Where to Go?

through a process of signal sequences, which are specific amino acid addresses that are recognized by cellular machinery

Protein Cotranslational Translocation

box 1: proteins are synthesised on poly ribosomes

• 16 - 30 NH  end Amino Acids - Hydrophobic 

• signal sequence

box 2: SRP - signal receptor particle 

•  6 proteins + 300 nucleotide RNA

• binds to the signal sequence

• protein synthesis stops

box 3: SRP/Ribosome/Nascent protein complex

• docks to the RER 

• SRP receptor

box 4: Nascent protein is transferred to the translocon and protein synthesis re-     commences 

•  SRP - recycles

box 5: signal peptide cleaves the signal sequence

box 6-8: Nascent protein is extruded into the RER lumen 

how to know the signal sequences is a RER specific targeting sequence?

A signal sequence is identified as an RER-specific targeting sequence because its presence on a nascent polypeptide chain causes the Signal Recognition Particle (SRP) to bind, temporarily halting translation and directing the ribosome to the Rough Endoplasmic Reticulum membrane for the protein's eventual translocation

Is there SRP?

protease - yes

Is GTP hydrolysis required for SRP recycling?

GMP - PNP - non hydrolyzable of GTP, GTP hydrolysis is required 

is there a signal peptidase?

yes

is the secretory protein totally inside the RER?

proteases - yes

how do we discover the translocon?

ohms law (V=IR)

identify translocaon?

photaffinity label

are multimeric proteins assembly in the rough ER?

in the RER - as this compartment is where protein folding, modification, and quality control take place before trafficking to other organelles like the Golgi apparatus.

what if something goes wrong in the RER? (proteins are abearent)

• activation of the UPR pathway

• attempt to resolve the stress by increasing folding capacity or, if unsuccessful, initiating Apoptosis (programmed cell death) and degrading the aberrant proteins via the proteasome.

Chaperones

• the proteins, which assist the covalent folding or unfolding and assembly and disassembly of other macromolecular structures

• monomers with a colecular weight of 70-100kDa

• most of them are heat shock proteins (HSPs)

• responsible for the folding, unfolding, assembly, and disassembly of proteins

ex: Dnak, DnaJ, GrpE, HtpG, and Hsp33

Chaperonins

• the proteins, which provide favorable conditions for the correct folding of denatured proteins, preventing aggregation

• oligomers with a molecular weight of 800kDa

• have a shape of two donuts stacked on top of one another to create a barrel

• responsible for the correct folding of denatured proteins, which prevent aggregation

ex: GroEL/GroES, TRiC

UPR (Unfolded Protein Response) pathway

• Recognizes mis-folded Proteins and Degrades Them

• Can Lead to Cell Death (Apoptosis) too under Severe Stress Conditions

• Tunicamycin – stimulates UPR by blocking N-linked glycosylation of nascent proteins (later)

• Salubrinal - Suppresses the UPR response

But how do RER Synthesized Proteins Become Integral Membrane (e.g. not secretory) Proteins with Different Topologies?

• through a co-translational process involving a signal recognition particle (SRP), the Sec61 translocon channel, and the protein's own amino acid sequence

• targets the ribosome to the RER membrane, where the nascent polypeptide chain begins to enter the Sec61 translocon channel

Type 1 single pass proteins

• SRP + SRP receptor

• signal sequences/peptidase

• stop and transfer anchor 

  • sequence - hydrophilic amino acids 

Type 2 single pass proteins

• SRP + SRP receptor 

• internal signal sequnces 

Tail advanced proteins

• aka tail (carboxy end) - anchored proteins

• RER

• No SRP + SRP receptor 

GPI (Glycosylphosphatidylinositol)

• a complex glycolipid that anchors proteins to the outer surface of the cell membrane

• n eukaryotes for roles like cell-surface receptors, enzymes, and adhesion molecules

Vesicular Traffic

• V-snare and T-snare - cofeis the specifity 

• COP II vesicles --> antergrade

• COP I vesicles --> retrgrade 

How about RER resident proteins? - POI, BiP - chaperone

• KDEL - 4 amino acids - present on resident protein 

• decrease in pH - is critical --> KDEL binds to the KDEL receptor --> returns home 

Dolichol Phosphate

• an RER Intermediate that can add N-linked Oligosaccharides in the RER

• sugars are removed

How important?

• N-linked oligosaccharide chains are processed en route

• involves initial trimming steps that serve as a quality control checkpoint for proper protein folding,

• followed by elaborate modifications that create the diverse structures necessary for protein trafficking, stability, and recognition in cell-to-cell communication.

CDG - Congenital Disorders of Glycosylation

Mutations in the Dolichol-linked
Oligosaccharide Biosynthetic Pathway

How about Glycosylation

• initatel in the RER

• continuous --> Golgi

• initial glycosylation is often due dolichol phosphate 

1. O-Linked - linked to serine --> shorter

2. H-Linked - linked to serine --> asynergie long + branched 

why glycosylation

1. increase protien varitey 

2. critical for folding --> function

3. protects proteins from extracellular environment 

How about mitochondria - how to study

in test tube

Are cytoplasmic factors involved (in the mito)?

yes - translocation

a. hsp70 - found in cytoplasm

b. hsp70 - also in mt matrix

are receptors required (in the mito)? 

yes 

how important is the mito targeting sequence (in the mito)?

:)

can folign proteins be translocated (in the mito)?

• no 

• experiment with DHFR (dihydrofolate reductase) and usually found in the liver

where does translocation occur (in the mito)?

at contact points

how about cholorplasts

• two pathways:

  • SRP pathay hdp70

  • pH dependent hsp70

Blood-Type-Altering Enzyme

• developed at the University of British Columbia, successfully converted a Type A kidney to the universal Type O

• offering hope to address the long wait times for Type O transplant candidates and potentially enabling universal donor blood for transfusions.

Peroxisomes

• targets sequence is the COO- end

1. proteins enter through nuclear pores, 30 proteins

2.  make < 40,000v  + nations - differs

3. others need to be imported 

4. imported as as FC + De

5. NLS = nuclear location signal impotrin

Secretory Pathways

• a cellular process that synthesizes, folds, and transports proteins for secretion outside the cell or delivery to specific organelles

• involves the movement of proteins from the endoplasmic reticulum (ER) to the Golgi apparatus, and to other locations

Golgi Apparatus

• many compartments:

1. condeasation of proteins

2. gycosylation

3. ADD sulfate + phiphots

4. proeolytic processing

5. cis-golgi (addres tag is added for lysosmal proteins)

Trans Golgi Netwrok (TGN)

• sorts proteins but not at first in the TEM (Transmission Electron Microscopy)

• last idenified in the RER pathway 

• MDCK cells on cell culture inserts --> typical phenotype 

• VSV (viscular ) G protein --> basal region

• HA glycoprotein --> apical region 

Constitutive Pathway

1. they are secreted as FAST as they are synthesized (ex: collagen)

2. immediatley sensitive to puromycin 

3. no electron vesicles (small-"lucient") 

Regulated pathway

1. proteins are stored in large vesicles - electron dense

2. point of secretion can be important

3. increase in Ca⁺²   is required for release

4. less senstive to puromycin"burst" of protein w/ outside signal 

How about lyosomes?

1. "zip code" - manwose -6-phosphete --> lyosomes

2. m6P is added cis-golgi

3. but, some lysosome/enzymes use the constitutive pathway

4. but - m6P receptor in the otuter plasma membrane