Vesicular Traffic, Secretion, and Endocytosis, part 2

Lecture 15

the trans-Golgi networks sorts

proteins into vesicles targeted for different destinations

lysosomal enzymes bear M6P residues that are

recognized by M6P-receptors and delivered by a clathrin-coated vesicle pathway to lysosomes

regulated secretory proteins are concentrated and stored

until secretion is signaled; constitutively secreted proteins are continuously delivered to the plasma membrane 

some proteins are processed into mature form after leaving 

the trans-Golgi network 

cargo not tagged for separatoin

secreted by default

model for formation of secretory vesicles

• included in vesicles when TGN fragments 

• plasma membrane proteins included in vesicles membrane

• densely packed secretary proteins concentrated by aggregation and trapped in vesicles 

exocytosis (secretion)

both constitutive (always on) and regulated pathways

constitutive provides cells with

newly made lipids and proteins to resupply/replace worn out membrane components as well as increase surface area for cell division 

also secrete extracellular proteins that will

become a parr of the extracellular matrix or diffuse to signal or nourish other cells 

proteins being constitutive secreted do not

possess a unique signal sequence

proteins whose secretion is regulated are

densely aggregated in the trans-Golgi network, allowing high concentration of secretion 

surface properties of such proteins promote

aggregation in response to low pH and high Ca2+ concentrations of trans-Golgi network 

regulated exocytosis pathway:

occur in cells specialized for secretion - such as hormone, digestive or neurotransmitter producing cells 

such secretions are stored in 

secretory vesicles near the plasma membrane and must wait for release signal

for example, excess of blood sugars triggers

insulin release from pancreatic beta-cells

trans-Golgi network sort proteins into

five different types of vesicles for transport to the plasma membrane, endosomes, and lysosomes (distal sorting compartment) 

if no oligosaccharide signal added in the Golgi,

the protein will be secreted from the cell

Five destinations: (1) COPI vesicles

retrograde transport of Golgi enzymes to the trans-Golgi (cisternal progression process) 

Five destinations: (2) AP complex vesicles (may have clathrin coat)

transport lysosomal enzymes directly to lysosomes

Five destinations: (3) Clathrin-coated (+AP2) vesicles

transport lysosomal enzymes to late endosomes for eventual delivery to lysosomes 

Five destinations: (4) constitutive secretory vesicles (unknown coat)

• transport constitutively secreted proteins and plasma membrane proteins to the plasma membrane

• cargo proteins include ECM proteins, blood proteins, immunoglobulins

Five destinations: (5) regulated secretory vesicles (unknown coat)

• store and process secreted proteins until signaled mot fuse with the plasma membrane to secrete the proteins

• cargo proteins include digestive enzymes and peptide hormones 

Arf GTPases assemble

clathrin coat

clathrin plays no part in

selecting specific cargo for the vesicles, their job is solely to bud the vesicle from the membrane (needs adaptor proteins)

the geometric association of clathrin proteins leads to 

spherical formation of the vesicle

once formed, clathrin in released from the vesicles, exposing 

receptor proteins that direct where the vesicle will be delivered 

the GTPase dynamic plays an essential role in

releasing the clathrin-coated vesicle from the membrane

the GTPase dynamin wraps around the membrane stalk and GTP hydrolysis causes 

conformational change that severs the membrane 

sugar residues added post-translationally to proteins in the Golgi complex can serve as an

“address label” to target proteins to the proper vesicles for delivery from the Golgi complex 

Golgi enzymes recognize

specific amino acids within the protein sequence

M6P targeting signal addition by

two cis-Golgi-resident enzymes ultimately leaving a 6-phosphorlyated mannose residue on the lysosomal enzyme

M6P residues direct

newly synthesized lysosomal enzymes to lysosomes

some proteins under

proteolytic processing after leaving the trans-Golgi

pro proteins matured into final form

after leaving the trans-Golgi

pro proteins include

soluble lysosomal enzymes; many membrane proteins, such as influenza hemagglutinin (HA); and secreted proteins such as serum albumin, insulin, glucagon, and the yeast alpha mating factor

several pathways sort membrane proteins to

apical or basolateral membrane regions of polarized cells with tight junctions

some cellular proteins - sorted similarly to

only apical (including GPI proteins in some cell types) or basolateral membranes 

some hepatocyte apical membrane proteins:

• sorted initially to basolateral membrane

• selectively endocytose into clathrin-coated vesicles and transcytosed through endosomes to the apical membrane 

specialized cells are able to internalize

large particles, even other cells

clathrin-coated pits generate

endocytic vesicles that are delivered to endosomes

in endocytosis, ingested material can

be recycled or sent to lysosomes

pinocytosis and receptor-mediated endocytosis are

clathrin-dependent 

phagocytosis 

• non-clathrin dependent

• cells and cell debris

• larger vesicles called phagosomes 

pinocytosis (Bulk-phase endocytosis)

• non-specific uptake of cellular fluids

• used for retrieval of membrane components 

receptor-mediated endocytosis

uptake of specific molecules

• bind to receptors in cell membrane

  • transmembrane proteins

  • binding site on exterior of cell

  • different receptors for different ligands 

in multicellular organisms, phagocytosis is used

infrequently for nutrient uptake (this is primarily accomplished by transporters and channels specific to certain molecules)

rather, in multi-tissues organisms phagocytosis is

restricted to cell of the immune system (white blood cells like macrophages and netrophils) that fight invasion by foreign microorganisms 

phagocytosis is used in single-cell protozoans as a 

feeding mechanism 

cell surface receptors recognize the foreign bodies either by 

the presence of specific molecules or because they have been tagged by host antibodies

polymerization of the cytoskeleton generates

cytoplasmic extensions called pseufopods that engulf the foreign body

coat-independent process:

fusion of membrane forms a phagosome that delivers the invader ultimately to lysosomes for degradation 

extracellular ligands bound to specific cell-surface receptors with cytoplasmic domain AP2-targeting sequences are

internalized by clathrin-coated vesicles

the endocytic pathway delivers some ligands (e.g., LDL particles) to

lysosomes, where they are degraded

the late endosomes acidic environment dissociated most receptor-ligand complexes for

receptor recycling to the plasma membrane and ligand degradation in lysosomes

the iron endocytosis pathways releases 

Fe3+ in the late end-some but recycles the transferrin carrier proteins with the receptor to the plasma membrane 

after internalization, vesicles-bound materials are transported in vesicles and tubules known as

endosomes

early endosomes are located

near the periphery of the cell

• it sorts materials and sends bound ligands to the late endosomes 

late endosomes are near the nucleus, also known as

multivesicular bodies (MVBs)

endocytic route

early endosomes

• located near cell periphery

• acidic pH 

  • maintained by H+-ATPase (V-type protein pumps)

• separation of ligand from receptor 

  • induced by acidic pH 

endocytic route: materials sorted

• house-keeping receptors recycled to cell membrane

• ligands and dissolved solutes transported to late endosomes

  • endosomal carrier vesicles formed from early endosomes 

endocytic route: late endosomes

• usually nearer to the nucleus

• destination of lysosomal enzymes included in Golgi vesicles

  • ligands are concentrated into late endosomes before transport to lysosomes for final processing

  • some receptors may be recycled to TGN (M6P receptor)

  • ubiquitin tagged receptors are transported to lysosomes for final processing 

endocytic route: lysosomal enzymes and endocytic material delivered to lysosomes

• several possible routes

  • maturation of late endosomes into lysosomes

  • fusion of late endosomes with lysosomes

  • transport for late endosomes to lysosomes in vesicles 

lysosomes

• small to relatively large

• spherical to irregular in shape

• bounded by single membrane

• heterogeneous interior 

functions of lysosomes 

organelles for cellular digestion in animal cells 

• acid hydrolyses for hydrolysis of almost every type of biological macromolecule

  • pH optimum in acid range

other functions of lysosomes

• degradation of ligands and dissolved macromolecules taken up by endocytosis

• digestion of solid materials brought into cells by phagocytosis

  • phagolysosome formed by fusion of lysosomes with phagosome

other functions of lysosomes - digestion of cellular organelles by autophagy

• organelle surrounded by membrane

• fusion of autophagic vacuole with lysosomes 

purpose of autophagy

• turnover of organelles during differentiation

• destruction of damaged organelles

• digestion of organelles during starvation

two types of receptors subjected to endocytosis

“house-keeping receptor” and “signaling receptor”

House-keeping receptor

responsible for uptake of materials that will be used by the cell

transferrin

mediates iron uptake

LDL receptors

mediate cholesterol uptake

House-keeping receptors generally lead to

delivery of the bound material to the cell and the return of the receptor to the plasma membrane 

signaling receptors responsible

for binding ligands that carry messages that change the activities of the cell

• hormones, growth factor, etc

receptor down-regulation -

internalization of signaling receptors usually leads to degradation of the receptor after signaling cascade is initiated

extracellular molecules bind

cell-specific receptor proteins — membrane anchored proteins that bind these ligands with high specificity: uptake of nutrients, metabolites, signaling molecules

receptor-mediated endocytosis: receptor in 

coated pits or move to coated pits 

receptor-mediated endocytosis - receptor binding

1. activates clathrin assembly

2. concentrated molecules to be absorbed

3. allows only cells expressing the receptor to absorb the molecule 

receptor-mediate endocytosis (RME):

used by cell to import specific macromolecules/complexes too large to be imported by membrane transporters 

• uptake specificity - receptor-dependent

• uptake mechanism - ligand-receptor complexes incorporated into clathrin/AP2-coated vesicles 

RME receptors

• some types cluster in clathrin-coated puts by cytoplasmic domain association with AP2 even in absence of ligand

• other types diffuse freely in the plasma membrane until a ligand-induced conformational change associated them with AP2

two or more types of receptor-bound ligand, such as LDL and transferrin, can be

present in the same coated pit

cells take up lipids from the blood in the form of

large, well-defined lipoprotein complexes

all classes of lipoproteins have the same general structure:

• shell composed of apoliprprotein and a phospholipid monolayer (not bilayer) containing cholesterol 

• hydrophobic core composed mostly of cholesterol esters/triglycerides (with minor amounts of other neutral lipids [e.g., vitamins])

LDL particle -

contains only a single molecule of one type of apolipoprotein (ApoB) wrapped around the outside of the particle

LDL receptor ar neutral pH (as at the cell surface):

• ligand binding arm seven cysteine-rich repeats (R1-R7) - tightly bind LDL apoB-100 (R4 and R5 - most critical for LDL binding)

• (Note: NPXY AP2-targeting sequence in receptor cytosolic domain)

LDL receptor at acidic pH (as in endosome):

• beta-propeller domain histidine residues - become protonated

• positively charged propeller domain binds negatively charged ligand-binding domain residues - causes release of the LDL particle

hypercholesterolemia (FH) patients have genetic defects in the

LDL receptor, resulting in increased circulating LDL and early development of atherosclerosis

RME endocytic pathway delivers

iron to cells without dissociation of the transferrin-transferrin receptor complex in endosomes

Apotransferrin

no found Fe3+

Ferrotransferrin

carries Fe3+ in blood

transferrin proteins bind to

transferrin receptor

endocytose membrane proteins targeted for degradation in the lysosome are 

incorporated into vesicles that bud into the interior of the endoscope 

cellular components (e.g., ESCRT_ that mediate endoscope membrane budding are used to

pinch off enveloped viruses such as HIV from the plasma membrane of virus-infected cells

autophagy envelopes a region of cytoplasm or an organelle into a

double-membrane autophagosome for delivery to a lysosome

most proteins targeted for a multi vesicular endoscope degradation - tagged with 

ubiquitin at the plasma membrane, in the trans-Golgi network, or in the endosomal membrane