D103 Golgi and Endo/Exocytosis (ALS 12, VIdeos 21 and 22)

where does the golgi secrete to

sorting depends on the sorting signal 

• cell exterior (secretion) 

• plasma membrane 

• lysosme 

• other parts of the endomembrane system

steps of protein trafficking

1. returned to the ER

2. retained in the Golgi apparatus

3. delivered to the endolysosomal system

4. delivered directly to the plasma membrane 

constitutive secretion

default path: proteins in plasma membrane and ECM, lipids, etc

• newly synthesized soluble proteins packed in new plasma membrane lipis and protein for unregulated membrane fusion

• delivers the soluble protein to the extracellular space

regulatory secretion

stored, wait for signal stimulation (ex - hormones, digestive enzymes)

• golgi releases secretory vesicle storing secretory proteins

• regulated membrane fusion is mediated via an intracellular signaling pathway (signal = hormone or neurotransmitter)

complications of constitutive transport

• unclear how the cargo is sorted into these carriers

• sorting signals not well defined (is traenport to the cell surface by default so that no sorting signal is necessary?) 

• difference to other transport: there is not a single dominant transport mech 

what does regulated secretion depend on

the release of cargo from storage granules

• stores hormones, mucus, and neurotransmitters

• sorting signal: proteins selectively aggregate with one another in TGN

• unknown sorting machinery

• exocytosis is triggered by a signal (usually chemical, like Ca2+)

lysosome

major degradation organelle

• diverse in shape and size

• fulfills variety of digestive functions

• very low pH due to ATP-driven proton pump

pH an proteins in the lysosome 

optimal pH = 5 

1. hydrolases that degrade other molecules 

2. cargo that is targeted for destruction 

digestive functions of lysosome

• breakdown of intra/extracellular material

• destruction of phgocytosed microorgs

• breakdown products serve a nutrient for the cell

niemann-pick disease

lysosomal storage disease

defects in the breakdown of lipids/cholesterol in the lysosome, leading to damage of brain, liver, and lungs

type A

• acid sphingomyelinase is affected (SM → Cer)

• SM accumulates in cells, leading to enlarged spleen and liver; also characteritic red cherry spot in the eye

• death in early childhood

how are proteins transported to the lysosome 

TGN → endosome (early, becoming late) → lysosome 

Step #1: mediated by clathrin-coated vesicles; transport cargo 

Step #2: maturation process where late endosome fuses with lysosome 

how are proteins transported from the tgn to the endosome

sorting signal: M6P

• a phosphorylated sugar that is part of the n-glycan (NOT a domain that is a part of the protein)

• generated in cis-golgi

receptor: M6P receptor

vesicle coat: clathrin

• 2 layers of the coat (like COPII)

• inner layer: adaptors → interact with the cargo

• outer layer: clathrin → membrane curvature

what regulates cargo-cargo receptor intearction in endosomal transport

regulated by pH

• determines the interaction between M6P and its receptor

• pH in TGN = 6.5 - 6.7 (M6P binds to receptor)

• pH in endosome = 6 (M6P no longer binds the receptor and dissociates)

overview of TGN to PM 

constitutive transport: mech not fully understood; multiple pathways 

regulated transport: aggregation of cargo to sort it into vesicle; fusion with PM in response to signal 

TGM to endosome transport

M6P serves as sorting signal

• not directly encoded by the primary AA of a protein

• requires a protein to be N-glycosylated in the ER; glycan is then moified to make M6P in the cis-Golgi → recognized by M6P receptor in TGN

M6P receptor bidnds to adaptor that in turn interact with clathrin coat

receptor-cargo interaction is regulated by pH → low pH of endosome promotes dissociation of cargo from receptor

M6P-R is recycled back to the TGN, buding of these retromer-coated vesicles is facilitated by the ER that “wraps” around the endosome

lysosome overview

membrane bound compartment that functions in degradation of proteins and also organelles 

• pH is lower (5) due to ATP-driven proton pump 

• contains many degradative enzymes 

proteins in this organelle

• function in the degradation of proteins (various hydrolases) 

• proteins that are delivered to be degraded → no return 

phagocytosis

cellular eating of large particles (microorganisms, senescnet and apoptotic cells) 

• actin-mediated process

• goal: delivery to the lysosome so that engulfed material can be destroyed 

pinocytosis

fluid phase endocytosis

• liquid and what is dissolved in it → non-specific

receptor-mediated endocytosis

goals:

• obtain nutrients (iron, cholesterol)

• remove potentially harmful molecules (neurotransmitters considered harmful bc signal may be lasting too long)

• remove signaling or adhesion molecules from the cell surface (allow cell to move forward)

mechanism: specific cargo selection via sorting signal

endocytic pathways that internalize specific cargo 

1. clathrin-dependent (most well-known)

• dynamin pinches off the vesicle; clathrin-coat falls off and fuses with the early endosome 

2. caveolin-dependent 

• forms around the vesicle and goes to the early endosome 

3. clathrin- and caveolin-independent internalization 

• vesicular or tubular → early endosome 

uptake of LDL cholesterol

example of receptor-mediated endocytosis

LDL receptor has specific endocytic sorting signal

separation of cargo and receptor due to low pH in endosome

1. activated small GTPase generates membrane domain with specific lipid composition

2. AP2 adaptor is recruited to this site

3. AP2 recruts cargo receptor, bound to cargo

4. AP2 also recruits clathrin coat → membrane curvature

AP1

tetrameric complex that moves from TGN → endosome

AP2 

tetrameric complex that moves from PM → endosome

comparison of the role of small GTPases in vesicle formation

COPII/I: Sar1/ Arf1 → coat (inner layer) → cargo (coat recrutis cargo)

clathrin: Arf1 → changes lipid environment in membrane → AP adaptor (=inner coat layer) → cargo, also bind to clathrin coat

• COP does not change the lipid environment

LDL and familial hypercholesterolemia (FH)

cholesterol is circulating in blood as lipoprotein particle (VLDL, LDL, HDL)

• LDL cholesterol is the “bad choleterol” because high levels of LDL promote plaque disposition → atherosclerosis and heart attack

• high LDL = high plaque = high MI rates

LDL uptake in FH pts

LDL uptake is defective

sorting signal in LDL receptor

• receptor cannot be recruited into vesicel

• LDL uptake is defective

AP2 adaptor

• sorting signal cannot be recognized

• ldl uptake is defective

normal LDL uptake

short cytoplasmic tail of LDL receptor interacts with the AP2 adaptor

• necessary and sufficient for uptake of LDL-R and its cargo LDL-cholesterol

fate of endocytosed receptors

determined by their sorting signals

LDL-R: recycled to cell surface

EGF-R (growth factor receptor): down regulation thru degradation by the lysosome

• early endosome mature into late endosomes

how are proteins sorted into the late endosome

sorting signal: mono-ubiquitin (polyubiquitin targets protein for degradation)

receptor to detect the signal: ESCRT-0

vesicle coat: none, but ECRT complexes help with the budding of the vesicle

EGF-R sorting signals 

1. clathrin-mediated endocytosis 

2. budding into the late endosome 

what does the fate of tm protein targeted to lysosome depend on

sorting signal

TM hydrolases: bisynthetic pathway; lack mono-ubiquitin sorting signal for budding into the late endosome → lysosome

TM proteins to be degraded: degradative pathway; contain the mono-ubiquitin sorting signals for budding into the late endosome → PM

autophagy

the lysosome can also degrade cytosolic proteins and organelles

goal:

• acquisition of nutrients: self-eating

• removal of non-functional organelles protein aggregates; microbes

• cellular mechanism fro degradation

steps:

• formation of crescent-shape cup (depends on Atg9)

• closure of membrane cup (autophagosome)

• autophagosome fuses with lysosome

uptake of material from the environment

phagocytosis: actin-mediated

endocytosis: pinocytosis/ receptor-mediated endocytosis

receptor-mediated endocytosis

• ex: LDL chol → receptors → uptake

• depends on a specific sorting signals in cytosolic domain of the receptor (TM protein)

• interact via adaptor protein (AP2, or monomeric adaptor) with clathrin

degradation of membrane proteins by the lysosome 

multivesicular bodies → vesicles that bud into the endosome 

sorting signal: mono-ubiquitin, machinery (receptor/coat): ESCRT complexes 

exocytic and endocytic pathways intersect at the endosome

exocytic route: transport of hydrolases that function in the lysosome

endocytic route: cargo to be degraded

degradation of cytosolic material

old organelles; autophagy