Folding, Assembly and Post-translational modification of Proteins

During insertion, nascent membrane proteins have to:

• adopt the correct orientation in the lipid bilayer
• undergo covalent modifications
• cleavage of the signal sequence
• N-linked glycosylation
• fold properly
• adopt their native state
• through interaction with ER-resident proteins such as chaperones

It is important to know that many of the proteins present in the lumen of the ER are in transit to other destinations. Others who are residents of the ER carry an "ER retention signal" composed of four amino acids at the C-terminus.

Two ER resident proteins are important:

1. protein disulfide isomerase (PDI) - catalyses the formation of disulfide bonds (S--S) from free sulfhydryl groups (SH) on cysteines
2. binding protein (BiP) - an hsp70-like chaperone that helps in translocation of proteins and also recognizes incorrectly folded proteins

Post-translational Modification of Proteins

Glycosylation

One of the major biosynthetic functions of the ER is the covalent addition of sugars to proteins. This is particular of proteins present in the lumen of the ER as very few proteins present in the cytosol are known to be glycosylated.

The process is carried out in the following steps:

• A precursor oligosaccharide is composed of 14 sugars, synthesized one sugar at a time and held in the ER lumen by a lipid molecular called dolichol 
• Transfer of the precursor as a whole to the side chain amino (--NH₂) group of the amino acid asparagine in the protein by oligosaccharyl transferase.

Hence this process is referred to as N-linked glycosylation. The energy for the transfer is provided by the high-energy bond of the pyrophosphate bridge that connects the first sugar in the precursor molecule to dolichol.

But what role does glycosylation play in the life of a protein?

Studies of two chaperone proteins calnexin and calreticulin were carried out which suggested a clue to the role of glycosylation in protein folding! Turns out that these chaperones bind incompletely folded proteins thus retaining them in the ER lumen and preventing them from reaching their destinations. 

The distintion between correctly folded proteins and incompletely folded ones lies in the fact that incompletely folded proteins have an affinity to the chaperones as those proteins undergo a continuous cycle of glucose trimming (by glucosidase) and glucose addition (by glycosyl transferase) at the terminal glucose site.  

Thus oligosaccharides can be thought of as "tags" that mark the status of protein folding.

Glycosylphosphatidylinositol (GPI) Anchor

Addition of lipids is known to direct the proteins to cell membrane. The ER enzymes covalently attach a glycosylphosphatidylinositol (GPI) anchor to the C-terminus of membrane proteins that are destined for the plasma membrane. 

Even after the translocation of the protein into the ER lumen is complete, the protein is held at the ER membrane by a hydrophobic C-terminal sequence of 15-20 amino acids. This sequence is then chopped off by an enzyme in the ER lumen and attachment of the protein to a pre-synthesized GPI precursor takes place. What is interesting is that this modification is specified in the C-terminal hydrophobic sequence itself!

Improper Folding and Misfolded Proteins

Improperly folded proteins are transported from the ER back to the cytosol and through the same translocon (Sec61 complex). Once in the cytosol, deglycosylation takes place by N-glycanase followed by ubiquitylation and eventual degradation in the proteasomes :(

Similar to the heat-shock response that is triggered upon the accumulation of misfolded proteins in the cytosol, accumulation in the ER triggers an unfolded protein response. This results in the increase of ER chaperones and other proteins associated with protein degradation. 

The unfolded protein response in yeast is thoroughly studied and involves transmembrane kinases, gene regulatory proteins and genes encoding ER chaperones.