Protein trafficking (golgi > lysosome/plasma membrane and endocytosis > lysosome)

COP I

coming out of Golgi and back to the ER in a retrograde fashion

*retrieval (KDEL)

COP II

leaves ER and is received in the cis-golgi (faces ER/nucleus)

Medial golgi

modifies sugar groups (enzymes do a lot of chemical rxns)

*mature

Trans golgi

sends off modified proteins

*mature

Clathrin

from extracellular space → trans golgi/endosomal compartments (backwards motion)

How does the cisternae obtain new enzymes @ each point in it’s progression?

retroactive shipping of compartmental enzymes to incoming stack (from ER) by COP I vesicles ensures new enzymes are obtained @ each point

Golgins

catch vesicles coming in and move across the golgi during maturation

Where are N-linked glycosylations added? how are they modified?

in the ER; modified in the Golgi (enzymes add a lot of sugar grps) as they move through the stacks →

1) complex - add NANA

2) adding MAN (high mannose)

Where are O-linked glycosylations added?

in the Golgi to the Ser or Thr residues (also modified in the Golgi - proteoglycans)

*as opposed to Asn for N-linked

Exocytosis pathways

in trans golgi

1) constitutive

2) regulated

Constitutively active pathway

no designated signal (always on), so Golgi sends proteins to the plasma membrane

*default

Regulated secretory pathway

involve proteins that are stored as aggregates near the membrane and wait for the release of the signal sequence

M6P

lysosomal sorting signal → in the form of a sugar instead of the usual aa sequences

*proteins w/ signal recognized by receptors in budding vesicles and bind → released in lysosome @ pH below 6

How does the functions of each cisternae create sorting signal for lysosomal transport?

cis: P-GlcNAC is added to the terminal mannose

trans: GlcNAC is removed, uncovering final M6P → recognized by M6P receptors in budding vesicles and bind @ pH 6.5-6.7

Lysosomes (structure)

enclosed membrane filled w/ soluble hydrolytic enzymes that digest macromolecules

*heterogenous in size/appearance → variety of functions and contents

Lysosomes (function)

clean up the cell/break down molecules

*H2O molecules break bonds w/in targets

pH of lysosome

acidic, stay b/w 4.5-5 → ensures the contents of the cytosol are protected should acid hydrolases leak out (compartmentalized)

*hydrolases work best @ acidic pH

How does the pH affect the delivery/activity of hydrolases?

lower pH = dissociation of hydrolase precursor (and M6P) from receptor

higher pH = binding of M6P to receptor

*V-type pumps use ATP to pump H+ in → low pH → attachment of M6P to proteins (necessary for lysosome function)

What is a consequence of a mutation in the active site of a single type of hydrolase (M6P can still be added and moved into lysosome)?

lipid accumulation in the lysosome, no beta-oxidation means it would look for other energy sources of lipid metabolism

What would be the consequence of a mutation in the catalytic/recognition site of GlcNAc phosphotransferase in the Golgi?

phosphorylated GlcNAc wouldn’t transfer to the mannose sugar on N-linked oligosaccharides → M6P signal wouldn’t uncover

• lysosomes wouldn’t contain acid hydrolases → unable to digest molecules

Endocytotic pathways

phagocytosis, pinocytosis, receptor-mediated endocytosis, macropinocytosis, autophagy

Phagocytosis

cellular eating using phagosomes → lrg particles, dead cells

(Ex. macrophages, neutrophils)

Receptor-mediated endocytosis

uses clathrin to bind macromolecules to complementary receptor proteins (enter the cell)

*import of cholesterol

Pinocytosis

cellular drinking, uses clathrin/caveolins (rate varies from cell to cell)

Macropinocytosis

clathrin-independent endocytosis (Ex. GFs, integrin ligands, apoptotic cell remnants) for degradation

Autophagy

self-eating; a cell disposes of obsolete parts (Ex. organelles)

• large macromolecules, protein aggregates, invading microbes

What do all endocytotic pathways have in common?

1) traffic to the lysosome and involve membrane/vesicular formation

2) involve retrograde movement

3) lysosomal directed

4) formation of internal endosomes

How is the receptor-mediated endocytosis mechanisms of LDL and iron similar?

• use clathrin pits

• fuse with/form endosome

• lysosomal directed

• require receptors to recycle to the surface

• pH around the 5-6

• endocytosis

How does an endosome facilitate LDL and iron endocytosis?

requires maturation in both pathways → pH adjustment, Rab remodeling, vesicle formation

*mediates process by transporting straight to the lysosome

How is the receptor-mediated endocytosis mechanisms of LDL and iron different?

in iron delivery → transferrin doesn’t dissociate from the receptor while in the endosome (pH shift allows iron to come off), empty transferrin recycled to exterior

What transport types are inhibited by a mutation in clathrin coat development?

pinocytosis, receptor-mediated endocytosis