Protein modification and trafficking

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There are two types of glycosylation

Protein Glycosylation

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a-amalyase is produced in the parotid gland and is found in saliva

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How glucose gets into cells  through the action of insulin

Protein Glycosylation

Where does Dolichol come from?

Dolichol is an isoprenoid compound synthesized by the same metabolic route as cholesterol. In vertebrate tissues, dolichol contains 18-20 isoprenoid units (90-100 carbons total). Dolichol is phosphorylated by a kinase that uses CTP to form dolichol Phosphate. Dolichol phosphate is the structure upon which the carbohydrate moieties of N-linked glycoproteins are built. After assembly on dolichol phosphate, the carbohydrate structure is transferred to an asparagine residue of a target protein having the sequence Asn-x-Ser/Thr, where X is any amino acid.

Cholesterol Biosynthesis

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Dolichol

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Disulfide bonds form between cysteines

	PDI protein disulfide isomerase works in the ER.  In the cytosol most Cystines are in the reduced state partly because of active oxygen radical scavengers.  In the ER PDI works by forming disulfide bonds with the target protein and then transferring that bond to another cystine within the target protein.

Further protein modification

	Why glycosylation?
		Aids in proper protein folding.
		Provides protection against proteases (e.g. lysosomal membrane proteins)
		Employed for signaling.
	Most soluble and membrane-bound proteins made in the ER are glycoproteins, in contrast to cytsolic proteins.
	Glycoprotein synthesis is a 3-part process:
			Assembly of the precursor oligosaccharide
			En-bloc transfer to the protein
			Modification of the oligosaccharide by removal of sugars

Where does glucose come from?

Starch is the major source of dietary glucose. The enzymes responsible for starch degradation are called amylases. Other sources of glucose are sucrose, a disaccharide glucose-fructose from fruits, and lactose, a glucose-galactose disaccharide from milk. Only monosaccharide species like glucose, fructose and galactose can be absorbed via active membrane transport systems. Special intestinal glucosidases split the disaccharides into their monosaccharide components. Maltose is hydrolyzed by isomaltase (oligo-1,6-glucosidase, E.C. 3.2.1.10) and, with lower efficacy, by sucrase (sucrose alpha-glucosidase, E.C. 3.2.1.48). Lactose intolerance comes from a lack of lactase in many adults, causing an accumulation of milk sugar with consequences such as dehydration.

Protein glycosylation

	Assembly of the precursor oligosaccharide…
	IMPORTANT POINTS:
		Assembly takes place on the carrier lipid dolichol, anchored in the ER membane.
		A pyrophosphate bridge joins the 1st sugar to the dolichol.
		Sugars are added singly and sequentially.
		After the two N-acetylglucos-amines are added, the assembly flips from the cytosolic side to the ER lumen.
		Nine mannose and three glucose molecules are added, totaling 14 sugars.

Now in the second step, attachment

	En-bloc transfer of the oligosaccharide to the protein
	One step transfer, catalyzed by oligosaccharyl transferase, which is bound to the membrane at the translocator.
	Covalently attached to certain asparagines in the polypeptide chain (said to be “N-linked” glycosylation).
	Attaches to NH2 side chain of Asn but only in the context:
			Asn-x-Ser  or   Asn-x-Thr

Finally modification of oligosaccharide

	Modification of the oligosaccharide by removal of sugars…
	Three glucoses and one mannose are removed sequentially in the ER.

Transport from the ER to Golgi

	Appropriately modified proteins leave the ER and travel to the Golgi Apparatus.
	They travel in membrane vesicles that arise from special regions of membranes that are coated by proteins.
	There are of three types of coated vesicles that are well characterized, clathrin-coated, COPI-coated and COPII-coated vesicles.
	COPI and COPII act mainly in ER or Golgi cisternae.
	Clathrin acts in Golgi or plasma membranes.

Clathrin coated vesicles

Clathrin cycle

Clathrin adaptins and dynamin

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Ubiquitin pathway for protein degradation

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Protein degradation

	For some proteins, more than 80% of peptides may not fold properly. These are removed from the ER and degraded.
	Retrotranslocation (or dislocation)
	Uses the same Sec61 translocator
	N-glycanase removes the oligosaccharide.
	Ubiquitin chain added to protein which marks it for degradation in the proteasome.

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