Function/Modification in Er/Golgi

Inserting proteins into ER membrane

Integral membrane proteins, hydrophobic alpha-helical transmembrane segments either single or multi pass

 

Type 1: Single pass N-term in Er lumen C-term in cytosol

Type 2: Single pass N-term in cytosol C-term in ER Lumen

Type 2:Multipass C-term in cyotsol

 

Membrane proteins have a N-Term Er Signal Sequence and an Internal stop transfer anchor sequence which form a hydrophobic alpha helix that remains in the membrane

• Er signal sequence is cleaved off

Domains can dissolve into lipid bilayer

Orientation is determined by the charge residues next to the transmembrane domain

• Proteins without a cleavable N-term ER signal sequence, it can have an internal hydrophobic signal anchor sequences

Multi-Pass protein integral membrane protein is determined by the change/orientation of the first transmembrane domain, The subsequent transmembrane domain must have the opposite charge/orientation

N-linked Glycosylation of ER proteins

Co-translational glycosylation

• Protein is glycosylated on the amide nitrogen of an asparagine residue, adding a pre-formed 14 unit oligosaccharide. 2 N-acetylglucosamine, 9 mannose and 3 glucose units

• Oligosaccharide is transferred by a lipid molecule dolichol to an asparagine residue with Oligosaccharyl transferase to speed up process

Processing of N-linked oligosaccharide

• ER processes and removes 3 glucose and 1 mannose residues

  • The 3 glucose being added is a signal for a mature oligosaccharide has formed

• Re-addition of 1 glucose residue has a role in protein folding

• O-linked sugars occurs in the Golgi (on serine and threonine hydroxyl groups

Folding polypep chains and subunit assembly

• Polypep is folds into final shape in ER lumen

• Carb binding protein Calnexin and Calreticulin (lectins) bind to incompletely folde proteins that contain 1 terminal glucose residue (hence re-addition)

• 3rd glucose removal causes chap. to dissociate from protein and leave ER

• BiP is a Hsp70 chaperone which bind to hydrophobic regions and prevent aggregation of poly pep

  • Prevent interaction of hydrophobic region of different proteins

• Will release poly pep by ATP hydrolysis, allows short opportunity to fold

  • If correct folding it is bured in the interior of the molecule

  • If incorrect folding it binds with BiP again

Disulfides Bond Formation

The enzyme PDI catalyzes disulfide bond in the lumen of the ER

• Disulfide bond in the active site of PDI is transferred to a protein

• PDI is an oxidizing agent (gets reduce and causes substrate to be oxidized) and must be re-oxidized by Ero1

• Bond only formed in Er lumen

Disulfide bond formation occurs between sequentially adjacent cysteines

Protein attachment to GPI anchors in ER

Protein is synthesized and inserted into ER membrane then a transamidase cleaves the precursor protein

• transfers the carboxyl group of the new C-terminus of the protein to the terminal amino group of the preformed GPI anchor

• The transmembrane domain gets a new N-term at the cleaved site

 Up Regulation of ER-based chaperones to relieve stress

UPR; unfolded protein response

Sensors are inactive by bonded to BiP. When there's accumulation of misfolded proteins, BiP is released to help fold leading to activation of the sensor ATF6 which moves to the golgi.

The cytoplasmic domain is cleaved from the transmembrane domain to fuse through the cytosol into the nucleus

It will stimulate the expression of genes encoding proteins that help alleviate the stress in the ER

• Chaperones

• Coat proteins to form on transport vesicles

• Proteins of quality control machinery

Up regulation of ER-based chaperones to stop protein synthesis

UPR

Release of BiP causes dimerization of PERK which becomes an activated protein kinase which will phosphorylate a protein/translation factor elF2a required for proteins synthesis

In phosphorylated state, eIF2a is inactivie and stops the cell from synthesizing additional proteins in the ER to give cell more time to process pre-existing proteins

Protein degradation machinery

UPR

Release of Bip from IRe1 causes it to oligomerize and brings its cytosolic kinase domains into close proximity with other cytosolic kinase domains to auto-phosphorylate and act as a ribonuclease

• It cleaves specific mRNA molecules which encode for transcription factors  for UPR target genes for ER molecular chaperones

Transport that move proteins out of the ER,

ERAD; ER-associated degradation

A misfolded protein is bonded to a chaperone and is transported into the cytosol.

N-glycanase removes N-linked mannose residues from N-linked oligosaccharides (also plays a part in targetting misfolded proteins to proteosomes)

Ubiquitin is a 76 amino acid protein that binds to lysine which targets the misfolded protein for degradation at the proteosomes.

Degrades protein into amino acid

Membrane Biogenesis

• Er is the source of membrane lipid like phospholipids and cholesterol

• Fatty acids for membrane phospholipis are synthesized in cytoplasm and incorporated into Er membrane on cytosolic side

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Establising Membrane Asymmetry

• Lipids can be transferred to lumenal side by phospholipid translocators (flippases)

• The type of phospholipid that is transferred across membrane is dependent on the translocator

Movement of lipids

• Organelles have enzymes that can convert between different types of phospholipids

• Some types of phospholipids are selectively included into vesicles

• ER can form contact sites with other organelles

  • Lipid transfer proteins can exchange lipids between the compartment allows for movement of lipids to outside endomembrane system

Additional Glycosylation occurs in the golgi

• Terminal glycosylation occurs in the golgi

• Modification by removal/additional of sugar side chains from the core oligosaccharide added in the ER

• Each step of glycosylation depends on the preceding modification within the golgi

• O linked glycosylation occurs here (adding oligosaccharde to OH on serine or threonine

• CIS GOLGI ADDITION OF PHOSPHATE AT 6TH CARBON OF MANNOSE IS AN IMPORTANT TAG FOR LYSOSOMES

Function

• Protein/lipid sorting at TGN

• Make glycoproteins/membrane more resistant to proteases by creating the glycocalyx

• Serve as cell recognition in cell-cell interaction

• Regulatory roles (protein folding/stability), ABO blood type and immune-recognition

ABO Blood Type

• ABO blood group antigens are carbohydrate structures added to lipid/protein on surface of RBC

Type A: N-acetylglucosamine (GalNAc)

Type B: galactose

Type O: unmodified

• Body creates antibodies for the opposite antigen

  • Type A will have antibodie for galactose

• Type O- is universal Donor (no antigens so acceptor bodies antibodies cannot bind)

• Type AB+ is universal acceptor (Has no antibodies)