Lecture 5: Vesicular transport

What does vesicular transport bring to our health?

Neuronal activities: dependent on vesicular transport
Blood vessel health: dependent on active endocytosis
Secretion of antibodies: immune responses
Secretion of digestive enzymes: exocytosis
Healthy eye: tears and moisture‐producing activities by exocytosis
Pathogens like SARS‐CoV2: often hijacking membrane trafficking machinery

What do pathogens like SARS-CoV2 do against vesicular transport?

hijacking membrane trafficking machinery

Overview of vesicular transport

1. enzyme synthesized in rough ER goes to PM

2. Cargoes are enclosed in vesicles

3. Specific vesicles carry specific cargo molecules

4. During transport, vesicles bud off from donor compartment and fuse with acceptor compartment

Secretory Pathway

outward from ER to Golgi and then other sites. Biosynthetic.

What are examples of secretory pathway?

ER → Golgi → Lysosome
Golgi → PM

Endocytic pathway

inward from the plasma membrane. Uptake.

Retrieval pathway

returning molecules belonging to their residing organelles. Recycling.

Jamieson & Palade Pulse-Chase experiment

• ³H-leucine (radioactive) is incorporated in the polypeptide

• System: pancreatic acinar cells: highly specialized cells for synthesis and secretion of digestive enzymes

• Assuming that chambers A, B, C and D are different compartments in the cell
  Feed cells and fix the cells by killing them.
  - label for short period of time and chase the cells
  Figure out the secretory pathway of ER → Golgi → secretory granules (vesicles)

R. Schekman Screening Yeast mutants defective for secretion

System: the budding yeast Saccharomyces cerevisiae
Isolating conditional (temperature sensitive) sec mutants defective in secretion at restrictive temperatures
Cloning the corresponding genes
investigating the function of corresponding proteins in vesicular transport

sec12

encodes a GEF protein required for vesicle formation in the ER
buildup in ER

sec 17

encodes a SNARE protein required for vesicle fusion with Golgi apparatus
- leaves the ER but can not enter Golgi

secretory mutant A

protein accumulates in the ER
ER X Golgi → secretory vesicles

secretory mutant B

protein accumulates in Golgi Apparatus
ER → Golgi X Secretory

secretory mutant C

protein accumulates in transport vesicles

Endoplasmic reticulum (ER): the starting point of the secretory pathway

• connected to nuclear envelopment

• ER in tubular and sheet‐like appearances
  Constantly being remodeled in living cells

How do proteins enter the ER? Co-translational:

1. growing polypeptide chain growing off mRNA

2. The ER-bound ribosome binds to the translocator of ER lumen.

Post-translational

1. free ribosome binds to chaperones

2. chaperones deliver to ER lumen

Microsome experiments

• human-made breakdown of ER membrane and homogenized

• rough and smooth microsomes are centrifuged with sucrose gradient concentration

smooth microsomes have a _________ and stop sedimenting at _______ sucrose concentration

low density, low

Rough microsomes have a _________ and stop sedimenting at _______ sucrose concentration

high density, high

Blobel & Dobberstein Protein translation experiment. How to learn whether proteins have been translocated into microsomes or not?

1. Label or probing the protein of intern

2. Separate microsome from others

3. Isolating proteins from the microsomes and the rest

4. Detecting the protein of interest

   *Proteins detected inside the microsomes have different size comparing the ribosomes.

Import into the nucleus

KKK-R-K

Export from the nucleus

M - 2 - 3 - Leu - 4 - 5- Leu - Ala -Ser -Ser -Phe

ER signal sequence

often cleaved so that mature protein becoming smaller than the one made by free ribosomes
1. signal sequence of growing peptide
2. Recognition: binding of SRP to ribosome causes translation to slow
3. Targeting: translation continues and translocation begins
4. Release: protein translocator
5. SRP receptor in rough ER membrane

Signal-recognition particle (SRP)

recognizing the ER signal sequence
- SRP: an assembly of proteins and RNA
- One of the proteins having a binding site for the signal peptide
- Ribosomes dock on SRP recognition by SRP receptor on the ER membrane

ER signal sequence with Signal-recognition particle (SRP) allows

translation-coupled translocation into the ER lumen

Soluble proteins delivered

Insertion of a membrane protein into the ER membrane

Start- and stop- transfer signals
1. transmembrane segment in SRP bound to protein translocator
2. signal petidase cleaves the signal peptide
3. translocator recieves the STOP signal to stop transfer into lumen but into lipid layer
4. mature single-pass transmembrane protein in ER membrane

How do cell know which side is facing the cytosol

charge of the amino acid on the edge of this transmembrane peptide

The positively charged side residues faces…

cytosolic side

The negatively charged side residues faces…

ER lumen

Insertion of multiple transmembrane domains of a protein into ER membrane

Alternating start- and stop-transfer peptide
Insertion into the membrane takes place during translation on the ER
1st start, 2nd stop, 3rd start

Rhodopsin

light-sensitive receptor protein found in the photoreceptor cells of the retina
7 transmembrane domains

Protein maturation inside ER lumen

• signal peptidase removes signal peptide at N-terminus of nascent polypeptide

• Glycosylation (e.g. N- linked glycosylation) starts in ER, finished in the Golgi

• N-linked glycosylation assists protein folding

What is the protein glycosylation site?

NXS/T
N-acetylglucosmine
mannose
glucose

You have a random amino acid following asparagine, but its always in the third position ________residue

[Ser - Thr ]

Folding of polypeptide inside the ER lumen

Disulfide bond formation between Cys residues
Chaperons, such as BiP, promoting protein folding and assembly: the chaperone
BiP retains incompletely assembled antibody molecules in the ER

Chaperons

BiP
keeps the incompletely assembled antibody molecules INSIDE ER

Polarized Golgi apparatus

Entry face: cis - phosphorylation of oligosaccharides on lysosomal proteins
Exit face: trans - suflation of tyrosines and carbohydrates SORTING → lysosome, PM, secretory vesicle

cis Golgi network (CGN)

forward or backward

trans Golgi network (TGN)

onward to destinations

Specialized Golgin proteins highlight

the membrane of Golig apparatus.

Each cisterna of Golgi contains…

different enzymes to catalyze modifications of oligosaccharide chains on proteins

Demonstrating of different cisternae contain different enzymes involved in glycosylation by…

immunogold labeling and electron microscopy

N-Linked Glycosylation

which carbohydrate molecules, known as glycans, are covalently attached to proteins, lipids, or other organic molecules. This process is crucial for the proper folding, stability, and function of many proteins and plays a significant role in cell-cell communication, immune response, and protein trafficking.

Occurs in the endoplasmic reticulum (ER) and Golgi apparatus.

Involves the attachment of a glycan to the nitrogen atom (N) of the asparagine residue in the protein.

This type of glycosylation starts with the assembly of a sugar chain on a lipid carrier molecule called dolichol phosphate, which then transfers the sugar chain to the target protein.

Why do proteins want to be glycosylated?

makes protein friendly to their environment and many proteins that are folded will need to be glycosylated to be recognized by bacteria

Glycoproteins often are found on…

plasma membrane
oligosaccharide chain facing the ER lumen earlier would be facing the extracellular space once inserted in the plasma membrane