1. Intracellular Compartments
The Endomembrane System

2. The Endomembrane System
A “continuous” System of membrane bound organelles:
Nuclear Envelope
Endoplasmic Reticulum
Golgi Complex
Endosomes
Lysosomes

3. The Endomembrane System
Endoplasmic Reticulum and Golgi Complex: Protein synthesis, processing and sorting
Early endosomes: sorting of materials brought into the cell by endocytosis
Late endosomes: Mature into lysosomes
Lysosomes: contain enzymes capable of breaking down macromolecules
Materials move between the endomembrane compartments through/by transport vesicles

4. The Endoplasmic Reticulum
Endoplasmic Reticulum (ER) a continuous network of flattened sacs, tubules, and vesicles.
The sacs are called cisternae, the space enclosed is the ER Lumen.
The ER is continuous with the nuclear membrane, so material in the ER lumen can move freely into the perinuclear space (between the two layers of the nuclear envelope)
Up to 90% of a cell’s membrane may be in the ER

5. The E R : Functions
Contains enzymes necessary for biosynthesis of proteins of the ER, Golgi, endosomes, lysosomes, plasma membrane
Biosynthesis of proteins secreted by the cell
Biosynthesis of lipids: triacylglycerols and cholesterol
The source of most of the lipids that are assembled into intracellular and plasma membranes

6. The E R
Smooth ER and rough ER: rough ER has attached (on the cytosolic surface) ribosomes
Rough ER is flattened sacs, smooth ER is tubules
Transitional elements: portion of the rough ER which forms transition vesicles (shuttle material to the Golgi): looks like smooth ER
Smooth and Rough ER are continuous one with the other: material can travel from one to the other

7. The E R
Cells involved in protein secretion (like the liver) have extensive rough ER networks
Cells involved in steroid production have extensive smooth ER networks
Homogenization of cells to purify subcellular components breaks the ER into small vesicles called microsomes: these have been important to studying ER function, but do not exist in the cell

8. The Rough ER
The ribosomes on the surface of the ER are responsible for synthesis of both membrane-bound (organelle and plasma membrane proteins) and soluble proteins (organelle and secreted proteins)
Most proteins that enter the endomembrane system enter the ER cotranlationally, that is, as translation is occurring
Most proteins of other membrane bound organelles (mitochondria, chloroplasts, peroxisomes) are transported there posttranslationally.

9. The Rough ER The rough ER is the site for:
Initial steps of carbohydrate addition (glycosylation)
Folding of proteins
Assembly of multimeric proteins
“Quality control”: improperly folded or modified proteins are retained or degraded

10. The Smooth ER The smooth ER is involved in: Drug detoxification
Carbohydrate metabolism
Calcium storage
Steroid biosynthesis
Membrane biosynthesis

11. Smooth ER: Hydroxylation Reactions
Hydroxylation reactions (addition of –OH group to an organic molecule) are important in drug detoxification (the hydroxylated form is more water soluble, and can be eliminated from the body) and in steroid biosynthesis.
These reactions depend on a cytochrome P-450 and NADPH
1) an electron transport system (in the smooth ER) transfers e- from NADPH to cytochrome P-450
2) Reduced cytochrome P-450 donates an electron to O2, activating it
3) O2 is reduced to H2O, -OH is added to the organic molecule

12. Smooth ER: Hydroxylation Reactions
Hydroxylation of fatty acids uses NADH (instead of NADPH) as an electron source
Enzymes which carry out these reactions (one atom of the O2 is added as a hydroxyl, the other is reduced to H2O) are called mixed-function oxidases, or monooxygenases
Mixed function oxidases can hydroxylate numerous compounds, and are upregulated by toxic compounds

13. Smooth ER: Hydroxylation Reactions
The behavior of oxidases explains several physiological observations:
Up-regulation (increased smooth ER) due to exposure to a toxic drug (example, phenobarbitol) results in habituation, larger and larger doses of the drug are required to achieve the same effect since the body is able to more efficiently degrade the drug.
The ability to act on multiple substrates explains the observation that exposure to one drug can reduce the effectiveness of other unrelated drugs (example, antibiotics in barbituate users)

14. Smooth ER: Hydroxylation Reactions
A different hdroxylation reaction, involving cytochrome P-448 and the enzyme aryl hydrocarbon hydroxylase, is involved in metabolizing polycyclic hydrocarbons: those containing two or more benzene rings (these are particularly toxic/ carcenogenic)
The oxidised products of this reaction are often more toxic than the original compound; aryl hydrocarbon hydroxylase can convert potential carcinogens into their active forms
Mice producing high levels of aryl hydrocarbon hydroxylase have high levels of spontaneous tumor formation
Cigarette smoke induces aryl hydrocarbon hydroxylase

15. Smooth ER:
Carbohydrate metabolism: The transfer of glucose from glycogen (in liver cells) in response to hormones (via the cAMP signal transduction pathway) is closely linked to smooth ER enzymes, such as the glucose-6-phosphatase, which removes the phosphate from glucose-6-phosphate (from glycogen breakdown) making it available for export (by a glucose transporter)
Calcium storage: smooth ER in some cells stores high levels of Ca+2, by using an ATP dependent Ca+2 pump: Ca+2 can be rapidly released when needed (such as activation of IP3 signaling pathway)

16. Smooth ER:
Biosynthesis of membranes: Most of the membrane phospholipids are made exclusively in the smooth ER
The enzymes involved in phospholipid synthesis are located on the cytosolic face of the smooth ER: phospholipids are added to the cytosolic leaf, then translocated (flipped) to the inner leaf by an enzyme.
Components of the endomembrane system get lipid by fusing to vesicles from the ER.
Mitochondria and chloroplasts (and the other membranes) get lipids by the action of phospholipid exchange proteins, which carry specific phospholipids from the ER, through the cytosol, to the organelle membrane.

17. Golgi Complex
The Golgi Complex is a series of compartments involved in protein modification, sorting, and secretion. The major divisions of the Golgi are:
Cis-Golgi network: closest to ER
Medial Golgi network: in the middle
Trans-Golgi network: farthest from ER, closest to cell surface
Transport vesicles

18. Movement Through the Golgi
Vesicles “bud” from the ER, fuse with the Golgi
Vesicles move from ‘layer’ to layer of the Golgi
Vesicles are moving in both directions, from ER toward cell surface and from trans-golgi toward ER

19. Golgi Processes Modification of N-linked oligosaccharides
O-linked glycosylation
Synthesis of oligosaccharides (such as extracellular matrix components)
Protein sorting: proteins are selectively transported ‘outward’ (anterograde) or ‘inward’ (retrograde). Signals in the protein and receptors in the membrane are involved in this process.

20. N-linked Glycosylation
This is the attachment of oligosaccharides to the amino group of certain asparagine residues in a protein.
There are two major steps: attachment of the “core oligosaccharide” and modification of the oligosaccharide.
Core glycosylation occurs in the ER.
Most of the modifications occur in various levels of the Golgi.

21. N-linked Glycosylation
Attachment of the core oligosaccharide occurs in the ER: the core is built on the ER membrane,. Attached to a lipid called dolichol phosphate.
After partial synthesis on the outside of the ER membrane, the glycosylated dolichol phosphate is flipped so the oligosaccharide faces the inside of the ER.
The Oligosaccharide is further extended, and then transerred to the –N of the glycosylation site of the protein.
Specific glucose and mannose residues are then removed from the core oligo in the ER.

22. N-linked Glycosylation
As the protein is carried through the Golgi, additional sugars may be added to the core oligosaccharide.
The modification pattern varies from one protein to the next, some proteins are not modified, others have extensive additions of N-acetylglucosamine, galactose, sialic acid, and fucose.

23. O-linked Glycosylation
Carbohydrate groups are also added to the –O of serine, threonine, and hydroxyproline residues as the protein passes through the Golgi.
One of the ways the movement of proteins through the golgi has been studied is by identifying the modifications occurring in various compartments.
Proteins can be recovered in the ER with modifications which are only carried out in the Golgi, demonstrating the retrograde motion.

24. Protein Sorting
Proteins contain specific sequences that signal to the cell’s machinery what the fate of the protein is to be.
For example, the sequence KDEL (lysine, aspartate, glutamate, leucine) at the carboxy terminus of a protein signals that it should be retained in the ER (not transported out).
However, some ER proteins are O glycosylated, indicating that they have been in the Golgi.

25. Protein Sorting
Retention of KDEL proteins appears to involve receptors for the KDEL sequence in membrane proteins of the Golgi.
Binding of the KDEL changes the conformation of the receptor, somehow triggering the membrane to incorporate into vesicles which are transported back to the ER.
Once fused with the ER, the pH is slightly more acidic than in the Golgi, which triggers a change in the Km of the KDEL sequence for the receptor, and the KDEL protein is released into the ER lumen.

26. Protein Sorting
Another method of protein sorting appears to depend on the length of membrane spanning regions of integral membrane proteins.
The thickness of the membrane increases as you move from the ER to the plasma membrane (from 5 nm to 8 nm)
There is a correlation between the length of the hydrophobic stretches of the membrane proteins and the thickness of the membrane of the Golgi compartment in which the protein ends up.

27. Protein Targeting
Proteins that move out of the Golgi must be targeted to end up in the right place (endosomes, lysosomes, secretory vesicles etc.)
Soluble lysosomal proteins are targeted by recognition of mannose-6-phosphate in the carbohydrate side chains by specific receptors on the membrane of trans-Golgi compartment (pH in trans-Golgi is about 6.4).
These receptor/protein complexes are then packaged into clathrin coated transport vesicles, and conveyed to an endosome.

28. Protein Targeting The endosome is called an early endosome.
As the endosome ‘matures’, the pH inside it decreases to about 5.5
At this low pH, the lysosomal proteins (mannose-6-phosphate containing) release from the receptor and become soluble in the endosome.
The receptors are recycled via vesicles that fuse to the Golgi.
The late endosome matures into a lysosome or fuses with a lysosome to deliver the contents (digestive proteins)