Cellular Endomembranes and Protein Transport: Lecture 2

What are the resident ER Proteins that assist in translocation and folding?

Binding Protein (BiP)

Protein Disulfide Isomerase (PDI)

Glycosyltransferases

What do resident ER proteins do?

They play a role in glycosylation, disulfide bridge formation and folding of newly synthesized secretory proteins.

Binding Protein (BiP)

An ER resident protein that "chaperones"; aids in folding and multimeric assembly, prevents back sliding, and unfolded protein response

Protein Disulfide Isomerase (PID)

An ER resident protein that catalyzes oxidation of free SH groups to disulfide bridges; forming and rearraigning disulfide bonds.

Glycosyltransferases

An ER resident protein that glycosylates proteins (N-link, O-link)

What is an O-linked oligosaccharide?

carbohydrate chains attached to the -OH group of serine and threonine

What is an N-linked oligosaccharide?

carbohydrate chains attached to an amide nitrogen of asparagine

Why are O-linked oligosaccharides required?

For glycoprotein expression and processing. They are critical for in vivo half-life and bioactivity.

Why are N-linked oligosaccharides required?

For their essential role in the folding and the quality control of proteins in the ER.

Biosynthesis of N-linked oligosaccharide precursor?

Dolichol phosphate (a hydrophobic lipid within the membrane) collects Carbohydrate groups from the cytosol using its phosphate group

What carbohydrate groups does Dolichol phosphate collects from the cytosol?

N-Acetylglucosamine (GlcNAc)

Mannose (Man)

How does Dolichol phosphate move the growing CHO chain inside the ER lumen?

because it is a lipid, it can flip flop within the cell membrane due to its fluidity

What is added to the precursor once it is inside the ER lumen?

glucose (Glc)

What is the composition of the completed N-linked oligosaccharide precursor?

3x Glc

9x Man

2x GlcNAc

When does N-linked oligosaccharide precursor synthesis take place?

before binding to the protein?

Where is the N-linked oligosaccharide precursor after it is synthesized?

In the ER lumen

What do the 3x Glc do on the N-linked oligosaccharide precursor?

They signal that the oligosaccharide is complete and ready to be transferred to a protein

Where does protein Glycosylation (N-linked oligosaccharide) take place?

On the Rough Endoplasmic Reticulum membrane.

Where do proteins go after protein glycosylation?

Proteins will be transported to the cis-golgi

How does protein glycosylation occur?

The ribosome performs co-translational translocation to make the protein while putting it inside the ER lumen.

The precursor and the protein attach.

Removal of 3x glucose and 1x mannose.

Quality control action.

Final protein + remainder of precursor leaves ER and travels to cis-golgi.

Does the ER lumen perform quality control on proteins before sending them to the cis-golgi?

yes; to ensure proteins are assembled properly

What is the final composition of the N-linked oligosaccharide once it leaves the ER lumen?

8x man

2x GlcNAc

What is and example of protein folding and assembly that we talked about?

Hemagglutinin; a glycoprotein found on the surface of some viruses/influenza.

What does hemagglutinin do for viruses?

the protein helps the virus attach to red blood cells, causing the cells to agglutinate

What is the function of BiP in the protein folding process?

BiP binds to exposed hydrophobic regions of the unfolded or partially folded HA.

This prevents HA from improperly interacting with other proteins or aggregating.

BiP then releases HA, allowing it to continue folding.

If HA is not folded correctly, BiP can bind and release repeatedly, giving it additional opportunities to fold correctly.

What is the function of calnexin and calreticulin in the protein folding process?

They bind to N-linked oligosaccharides, preventing incorrect folding by preventing aggregation of adjacent protein; helps glycoproteins fold correctly

Which type of membrane protein is involved with Hemagglutinin folding and assembly?

Type I

- specifically 3 Type I proteins

What happens if protein misfolding occurs?

The unfolded-protein response with Ire1 and Hac1

or

Retention of oligosaccharides with CNX/CRT and OS-9

What activates Ire1 during ER stress?

Accumulation of misfolded proteins in the ER lumen.

What happens to Ire1 when misfolded proteins accumulate?

Ire1 binds monomer binds to BiP.

Ire1 monomers dimerize, activating its endonuclease activity and cutting Hac1 mRNA.

What is Ire1's main function?

Ire1 cuts and splices unspliced HAC1 mRNA.

What happens to HAC1 mRNA after Ire1 cuts it?

The mRNA is spliced, moves back to the nucleus, and can be translated into Hac1 protein.

What is Hac1?

A transcription factor produced from spliced HAC1 mRNA.

What is Hac1's main function?

Hac1 enters the nucleus and activates genes that help resolve ER protein misfolding.

How does Hac1 help solve ER protein misfolding?

It increases transcription of genes involved in protein folding and ER quality control.

What is the overall Ire1 → Hac1 response?

Misfolded proteins → Ire1 activation → HAC1 splicing → Hac1 → folding and quality-control genes.

What is the starting N-linked oligosaccharide?

Glc₃Man₉GlcNAc₂ (3 glucose, 9 mannose, 2 GlcNAc).

What is the function of CNX/CRT?

CNX/CRT binds the one-glucose form and retains the protein for folding.

What does UGGT do?

UGGT adds one glucose back if the protein is misfolded, allowing CNX/CRT to bind again.

What happens to a protein that remains misfolded?

ER α-mannosidase removes mannose residues, allowing OS-9 to recognize it.

What is the function of OS-9?

OS-9 recognizes persistently misfolded proteins and targets them for ERAD and degradation.

What is the key difference between CNX/CRT and OS-9?

CNX/CRT = retain and refold; OS-9 = target for degradation.

What are the names of the three parts of the golgi?

Cis, Medial, and Trans

What is endocytosis?

the taking in of matter by a living cell by invagination of its membrane to form a vesicle

What can vesicle trafficking do?

govern the composition of the plasma membrane by taking in proteins.

What are the three regions of the Golgi?

Cis-Golgi, medial-Golgi, and trans-Golgi.

What is the direction of protein transport through the Golgi?

Proteins generally move from cis → medial → trans Golgi.

What is endocytosis?

The process of bringing material from outside the cell into the cell in vesicles.

Where does retrograde transport occur?

Retrograde transport moves cargo backward, from later Golgi compartments to earlier ones and from the ER back to the ER.

What is the difference between anterograde and retrograde transport?

Anterograde moves forward; retrograde moves backward to earlier compartments.

What is the mechanism by which vesicles are formed?

Budding and fusion

What are the three types of coated vesicles?

COP II, COPI, and Clathrin

Where does COPII travel?

from ER to cis-golgi

What are the coat proteins associated with COPII?

Sec23/Sec24 and Sec13/Sec31 complexes, Sec16

What type of GTPase is COPII associated with?

Sar1

Where does COPI travel?

Carry proteins from cis-golgi to the ER

and

Transport proteins from later golgi cisternae back to earlier golgi cisternae

What coat proteins is COPI associated with?

coatomers containing seven different COP subunits

What GTPase is COPI associated with?

ARF

Where do Clathrin and adapter proteins travel?

from trans-golgi to endosome

and

plasma membrane to endosome

and

golgi to lysosome, melanosome, or platelet vesicles

What coat proteins are associated with Clathrin/adapter proteins?

Clathrin + AP1 complexes, GGA, AP2 complexes, AP3 complexes

What GTPase is associated with Clathrin/adapter proteins?

ARF

What activates Sar1 and starts COPII assembly?

Sec12 exchanges GDP for GTP on Sar1, activating it and attaching it to the ER membrane.

What happens after Sar1-GTP binds the ER membrane?

Sar1 recruits Sec23/24, causing the COPII coat to assemble and the vesicle to bud.

What causes COPII coat disassembly?

Sec23/24 promotes Sar1 GTP hydrolysis; Sar1 becomes GDP-bound, causing the coat to disassemble.

True/False: ARF plays a role in coat formation of COPI and Clathrin

True: ARF is GTPase for COPI and Clathrin

What are the molecular signals on vesicles that cause them to bind to only the appropriate target membrane?

Targeting signal sequences

T/F: different types of vesicles have different sorting signals

true

Di-acidic signal sequence

Signal Bearing protein: cargo membrane proteins in ER

Proteins with signal: COPII Sec24 subunit

Signal Receptor: COPII

KDEL signal sequence

Signal Bearing protein: ER-resident soluble proteins

Proteins with signal: KDEL receptor in cis-golgi membrane

Signal Receptor: COPI

What is the main function of Rab-GTP in vesicle transport?

Rab-GTP helps the vesicle dock at the correct target membrane by binding a Rab effector.

What are v-SNAREs and t-SNAREs?

v-SNAREs are on the vesicle; t-SNAREs are on the target membrane.

How do SNAREs cause membrane fusion?

v-SNAREs and t-SNAREs form a SNARE complex that pulls the two membranes together, causing fusion.

What does NSF do after membrane fusion?

NSF uses ATP hydrolysis to disassemble the SNARE complex so the SNAREs can be reused.

Forward protein trafficking (anterograde)

Done by COPII

Delivers protein from ER to cis-golgi.

Takes SNARE proteins to the golgi.

Reverse protein trafficking (retrograde)

Done by COPI.

Retrieves v-SNAREs and other materials back to ER for the next forward transport.

Takes missorted BiP and PDI back to ER.

What protein trafficking vesicle is associated with a Di-acidic sorting signal?

COPII performing anterograde transport

What protein trafficking vesicle is associated with a KEDL sorting signal?

COP1 performing retrograde transport

How does pH affect KDEL?

Low pH = promotes binding between KDEL and its receptor

High pH = low binding between KDEL and receptor; releasing peptides in the ER.

What is included in the early stage of the secretory pathway?

Transport by COPII and COPI between the ER and Golgi