MBB231 - Lecture 13: Vesicular Transport II

explain the addition of mannose 6-phosphate to targeted proteins destined for the lysosome

• enzyme (N-acetylglucosamine phosphotransferase) takes a phosphate group with a sugar (GIcNAc) → sticks to mannose sugar on the lysosomal protein

• protein has a phosphate tag - still covered by GIcNAc

• second enzyme (phosphodiester glycosidase) snips off the GIcNAc sugar

• mannose 6-phosphate tag - exposed and active

How does the M6P receptor dissociate from M6P in the late endosome?

dissociates through acidic pH → results in the conformation change of the receptor protein in the late endosome

what is the enzyme deficiency / material accumulated for inclusion cell disaese (I-cell disease / muclipidosis II)

enzyme deficiancy: lysosomal enzymees

material accumulated: glycolipids, glycoproteins, sialyloligosaccharides

what is constitutive secretion (exocytosis)

• after budding from TGN (trans golgi network) → vesicle moves directly to the cell surface → immediately fuses with the plasma membrane

• unregulated

• continuous

• independent of external signals

what is regulated secretion (exocytosis)

secretory vesicles accumulate in the cell → only fuses with the plasma membrane in response to specific signals

what is the general process for regulated secretion of exocytosis

• immature regulated secretory vesicles bud from the TGN

• secretory proteins become mature through condensation (+ sometimes proteolytic processing)

• mature secretory accumulate by the site of secretion

• membrane fusion is initiated by a hormonal / chemical signal

what is polarized exocytosis

• where transport vesicles (from Golgi / endosomal compartments) are directed to → fuses with a specific targeted domain of the plasma membrane

• vesicles have receptors that bind to the localized sites on the plasma membrane

• temporally regulated (controlled/adjusted with respect to time) → to coordinate process and distribution with development and physiological events

what is exocytosis

proteins in vesicles go to exterior of the cell when the vesicle fuses - proteins can be added to the membrane

what is endocytosis

essential nutrients from the extracellular space are ingested by the cell → vesicles bud inwards from the plasma membrane - proteins / lipids can be removed from the membrane

what is the bulk phase (fluid phase)

→ helps in maintaining membrane + fluid balance

• process is non-specific

• does not require any special clathrin proteins to form the pocket

• material trapped in vesicles reflect it’s concentration in the extracellular space → higher material concentration in the extracellular = more brought in into the cell

• occurs at a constant rate

• compensates for the addition of proteins/lipids on the plasma membrane from exocytosis

what is receptor mediated endocytosis

→ used to selectively + efficiently acquire macromolecules

• cells use receptors on the outer cell surface to internalize macromolecules

• each receptor recognizes a different macromolecule

what is the pathway of receptor mediated endocytosis

• ligands bind to their receptor on outer cell surface

• complex (ligand + receptor) diffuse laterally → encounters specialized regions - coated pits (site for collection / internalization of the complexes)

• accumulation of complex in the coated pit → triggers the accumulation of Clathrin-coat proteins on cytosolic side of the membrane

• induces a curvature → invagination (membrane folds inward upon itself)

• pit will pinch off forming a coated vesicle

how are clathrin-coated vesicles differ from the COPII vesicle

differs in their formation and proteins → clathrin-coated vesicles extensively overlap while COPII do not

what does each clathrin molecule consist of

• 3 heavy chains

• 3 light chains

→ join at the centre to make a triskelion

what does the adaptor protein (AP2)

promotes the formation of the clathrin cage + recruits membrane receptors to the budding vesicle

what does phosphoinositide (PI [4,5]P₂) do to adaptor protein 2 (AP2)

binds to AP2 - resulting in a conformation change → make the cargo binding site accesible

what is dynamin required for - how is it used

• as the clathrin accumulates around the budding vesicle - dynamin is needed in order to constrict and close the vesicle

• dynamin is a cytosolic GTPase - when GTP is hydrolyzed - dynamin rings righten → inducing constriction to separate the vesicle from the plasma membrane

what are the structural components of LDL

• amphipathic shell - phospholipid monolayer

• contains cholesterol

• single molecule of protein - ApoB

• non-polar core contains many cholesterol esters and triglycerides

what is the process of receptor mediated endocytosis of low density lipoprotein (LDL)

• cell surface has LDL receptors that bind to the ApoB protein on the outer phospholipid monolayer of the LDL particle

• NPXY sorting signal on the cytosolic tail of the LDL receptor interacts with the AP2 adaptor complex → forms the clathrin coated pit on the plasma membrane

• clathrin coated pits are pinched off by dynamin

• clathrin coat is shedded - vesicle fuses with the late endosome

• late endosomes fuses with the lysosome → protein / lipids are broken down

• receptor recycles to the surface - ready to bind to another LDL particle

how does the cell use acidity to intake cholesterol and drop it off inside the cell

at cell surface ( pH is approx. 7 )

• LDL receptor acts like a grabbing arm

• ligand binding arm is negatively charged → tightly grasps onto LDL particle

→ cell pulls the receptor + LDL particle into an endosome

inside the endosome (pH is approx. 5)

• inside of the endosome becomes acidic

• acid adds positive charges to a part of the receptor - beta-propeller domain

• receptor releases the LDL particle and binds to the beta propeller instead (opposite charges attract)

what are some examples of phagocytosis

single celled organisms → use phagocytosis to eat food

humans (immune cells) → white blood cells (i.e. macrophages / neutrophils) use phagocytosis to swallow and destroy invaders (i.e. germs)

how does phagocytosis work

• cell’s outer membrane reaches out using pseudopods (arm like extensions) → wrap around the target

• membrane pinches off - target particle is trapped in a phagosome

• phagosome merges with endosomes (late endosome + late phagosome) → lysosomes (with digestive enzymes) → breaks down and destroys the target particle

what is the difference in endocytosis process for housekeeping receptors and signalling receptors

housekeeping receptors - are recycled

• receptor delivers bound material → early endosome

• releases material due to low affinity for the material in low pH (in early endosome - hydrogen ATPase pumps hydrogen into the endosome)

• empty receptor is pinched off into recycling compartments → sent back to the cell surface to bind to more material

signal receptors - are degraded

• binds to extracellular messengers that change the activity of the cell depending on the message

• is degraded (down regulation) to reduce the cells sensitivity to the stimulation → reduces problems that can occur (i.e. cancer)

• are packed into endosomes → travel down to late endosomes → fuses with lysosomes where digestive enzymes break down the signal receptor + message