endocytosis 2

pathways taken by endocytosed cargo

• LDL

• EGF and its receptor

• iron

• transcytosis

• endothelial transcytosis

• phagocytosis

endocytosis of LDL

• LDL particle binds to its receptor on plasma membrane

• internalized via clathrin-dependent endocytosis

• LDL-receptor complex dissociates in early endosomes due to mildly acidic environment

• receptor returned back to plasma membrane

• LDL particle degraded in lysosome → cholesterol, amino acids, fatty acids

endocytosis of EGF and EGFR

• EGF binds to its receptor and induces endocytosis

• internalized EGF-EGFR complexes are stable in early endosomes

• EGFR inactivated by sequestration in intraluminal vesicles in multivesicular body

iron in blood

• Fe(II) (ferrous iron) versus Fe(III) (ferric iron)

• blood plasma iron travels as Fe(III)

• Fe(III) binds to glycoprotein transferrin

• two forms: apo-transferrin and holo-transferrin

• most plasma iron taken up by reticulocytes

apo-transferrin

• not bound to iron

• comes off receptor at neutral pH

holo-transferrin

bound to iron

endocytosis of iron

• two holo-transferrins bind to receptor on reticulocyte surface

• holo-transferrin-receptor complex internalized via clathrin-dependent endocytosis

• in early endosomes, Fe(III) released from transferrin

• Fe(III) reduced to Fe(II) by STEAP3, then transported into cytoplasm by transporter DMT1

• apo-transferrin-receptor complex recycled back to plasma membrane

• neutral pH causes apo-transferrin to dissociate from receptor

transcytosis of immunoglobulins

• couples endocytosis and exocytosis

• transports cargo from one side of cell to another

• immunoglobulins transported by receptors

  • secretory IgA → pIgA

  • IgG → FcRn

transcytosis of IgA

• IgA mainly in mucosal tissue

• plasma cells secrete dimeric IgA

• pIgA at basolateral region of epithelial cells binds to IgA

• complex is clathrin-dependent endocytosed, travels to apical side of cell

• at apical side, pIgA is cleaved (TM still with cell, extracellular with IgA)

• sIgA released into lumen

caveolae-mediated transcytosis

• involved caveolae-mediated endocytosis

• separate from standard endocytic pathway

lipid rafts

• lipid microdomains

• enriched in cholesterol and sphingolipid

• assembled at Golgi

• compartmentalize proteins, accommodate special TM proteins, GPI-APs (glycosylphosphatidylinositol-anchored proteins), signaling molecules

caveolae

• small flask-shaped pits in plasma membrane

• coated in caveolin, integral membrane protein

• concentrated cholesterol-rich membrane

• concentration of some signaling molecules

endocytosed caveolae

• can fuse to form neutral pH compartments called caveosomes which are distinct from endosomes

• one role appears to be transcytosis of albumin and other proteins across endothelia

  • very rapid process

  • appear to pinch off from side facing capillary, cross cell, fuse with plasma membrane facing tissue

non-endocytosed caveolae

• may function as signaling platforms, as many signaling molecules on both leaflets of plasma membrane prefer raft-like membranes

• may serve to increase effective local concentration of some signaling molecules and increased efficiency of signaling

transcytosis of albumin

• binds to gp60 at apical endothelia → activates transcytosis

• gp60 associates with caveolin → caveolae form

• dynamin dependent

caveolar-dependent endocytosis

• caveolae found in most cells

• lipid rafts preferentially associate with caveolin

  • caveolin-1 binds to cholesterol

• caveolin-1: major component of caveolae

• other proteins for shape and function → cavins, Pacsin2

• internalized compartment called caveosome

  • neutral pH, caveolin-1 positive

caveolae as signaling platforms

• normally static structures

• can sit on surface for a long time without endocytosis

• some signaling molecules bind to conserved caveolin-scaffolding domain (CSD) on caveolin-1

• enriches signaling molecule at membrane

vesicular intra-Golgi trafficking model

• cisternae are fixed

• COP-I vesicles move between cisternae

• vesicles do not transport cargo, but instead Golgi enzymes

cisternal maturation intra-Golgi trafficking model

• no transport intermediate

• cargo stays in cisternae

• evidence in algae and mammalian cells

• enzymes need to be constantly relocating to remain in right place

direct evidence for cisternal maturation in mammalian cells (experiment 1)

• procollagen only exits ER in presence of ascorbic acid (vitamin C)

• add ascorbic acid for short time to let procollagen exit ER, then remove

• procollagen appears first in cis-Golgi, then in medial compartments, then in trans

• it is not spread over entire Golgi → cisternae are polarized

direct evidence for cisternal maturation (experiment 2)

• ts045 mutant of VSVG misfolds at 40°C and is held in ER by quality control machinery (CNX, CRT)

• it can still fold correctly and leave ER if cell is shifted to 35°C

• once out of ER, it will continue through secretory pathway regardless of temperature

• can be allowed to leave ER for short period of time, then go back to 40°C

• found in cis-Golgi → medial → trans

possible explanations for movement of Golgi enzymes between maturing cisternae

1. COPI vesicles move enzymes backwards at same rate as cisternal maturation

   • appear to be different variants of COPI vesicles, but it is difficult to see how they can be fine-tuned to always put the enzyme in the right place

2. enzymes move within Golgi (perhaps randomly) and are localized by other means such as selective affinity for their substrates

   • resident proteins typically have shorter transmembrane domains than cargo integral membrane proteins, so they may prefer to be in different lipid domains

COPI for retrograde trafficking

• required for recycling of proteins from Golgi to endoplasmic reticulum

• proteins that cycle between ER and Golgi (particularly cargo receptors) are highly concentrated in COPI pits and vesicles on both Golgi and VTC

• these proteins have COPI interacting sequences on their cytoplasmic portions

• deletion of COPI subunits in yeast lead to appearance of cargo receptors on cell surface

intra-Golgi trafficking in 2020s

• cisternal maturation is now known to be how cargos are transported through Golgi apparatus

• cisternal maturation does not explain how different Golgi enzymes are kept segregated in separate cisternae

• both other models, direct connections between cisternae, and shuttling of Golgi enzymes by COPI vesicles still have supporters