Bio 225 - topic 4

Endomembrane system

Comprised of ER, golgi, endosomes and lysosomes (does not include peroxisomes)

Endoplasmic reticulum

Proteins destined for plasma membrane, organelles and exports biosynthesis of lipids

free ribosome

translation takes place in cytosol, completed polypeptide protein can: remain in cytosol, travel to ER, nucleus, mitochondria, chloroplast or peroxisome

bound ribosomes

Once the polypeptide chain is created, it either remains in the ER, or travels to the golgi. After the golgi: secretory vesicle, lysosome, transport vesicle to plasma membrane

Signal mechanism of ribosomes that get bound to ER (co-translation/translocation):

The SRP recognizes and binds both the signal sequence and the large ribosomal subunit, translation stops momentarily. Docking of the ribosome to the ER membrane occurs by 2 key interactions…one between the SRP and the SRP receptor and the other between the ribosome and the translocon. SRP and SRP receptor each bind a molecule of GTP…triggers the restart of translation, opening the pore, and the insertion of the signal sequence into the pore. GTP hydrolysis results in release of the SRP, During translation the signal sequence is typically removed by a signal peptidase and degraded. The result is a protein that is now a soluble protein within the ER lumen; further processing typically occurs, including glycosylation and various modifications that may occur in the ER or golgi. N-terminus = first part of protein that exits ribosome

Polypeptide with internal stop-transfer sequence and terminal ER signal sequence

Stop transfer sequence halts process of translocation, polypeptide moves out through a side opening in the translocon, anchoring in membrane. this creates a transmembrane protein with the C terminus in cytosol and N terminus in ER lumen (C terminus is found inside the ribosome during translation)

Polypeptide with a single internal start-transfer sequence

Starts polypeptide transfer and then moves through a side opening in the translocon to anchor itself in the membrane. the N terminus is in the cytosol and the C terminus is in the ER lumen. if this polypeptide also had a stop-transfer sequence that prevents complete transfer of the polypeptide through the translocon, both the N and C terminus would be in the cytosol

N-glycosylation

Most proteins made at the RER are N-glycosylated in a cotranslational manner by oligosaccharyltransferase, a transmembrane protein complex of the RER. All proteins receiving and N-linked oligosaccharide are given the same "core oligosaccharide".(Oligosaccharide - chain of carbohydrates). Oligosaccharide added by an oligosaccharyltransferase to an N in Asn (asparagine) that is part of the signal sequence Asn-X-Ser/Thr (X = any amino acid but proline and glycine). Core oligosaccharide is modified in the ER and/or Golgi

Smooth ER

Involved in lipid metabolism, production of lipoprotein particles (carry lipids via bloodstream to other parts of body) and detoxification of lipid soluble drugs and other harmful products (cytochrome p450). Transition ER are areas of the smooth ER which form transport vesicles for delivery to the golgi. Important for intracellular Ca2+ storage

Drug detoxification

Often takes place in liver cells, hydroxylation often the first step, increase solubility and introduces a site for further modification

Oxidation

P450 in liver plays a major role

Carbohydrate metabolism (SER of liver cells)

Glucose-6-phosphate localizes to SER, involved in releasing glucose from glycogen storage molecule, allows glucose to leave liver

Calcium storage (SER of liver cells)

Pumped into SER by ATP-dependent calcium ATPases, important for signalling

Steroid biosynthesis (SER of liver cells)

Biosynthesis of cholesterol and steroid hormones

ER and lipid biosynthesis

Most lipid biosynthesis enzymes exclusive to the ER. this means most phospholipids and cholesterol are manufactured on cytosolic face of ER, requires flippases, even distribution and asymmetry, moved by endomembrane system and cytosolic exchange proteins (phospholipid transfer proteins)

Golgi

Cis face oriented towards ER, trans face away from ER, compartments are biochemically and functionally distinct

stationary cristrnae model

Cisternae stay stationary, vesicles break off between cisternae

Cisternal maturation model

Cisternae rotate, new vesicles come in (from ER) and create new cristae, resident proteins are changing

anterograde transport

movement from the ER through the Golgi towards the plasma membrane

retrograde transport

the flow of vesicles from the Golgi back towards the ER, (these processes even out the overall membrane balance)

Role of golgi in glycosylation

N-linked glycosylation initiated in the ER, as proteins move through the golgi, N-glycosyl group is modified, contains many glucan synthetases and glycosyl transferases, each process is mediated by different enzymes

Challenges in protein trafficking

Proteins localize to ER, golgi, endosomes, lysosomes, plasma membrane and extracellular space, proteins utilize a variety of tags for sorting, membrane lipids may also be tagged to direct trafficking of vesicles (golgi - directs where things go)

Retaining/retrieving ER proteins

Resident ER proteins are either retained or retrieved, kin recognition may allow proteins to be retained, retention tags may also keep proteins in the ER, in contrast, retrieval tags such as KDEL and KKXX may be used to return ER proteins

Golgi complex proteins

Resident golgi proteins are integral membrane proteins with one or more transmembrane regions, membrane increases in thickness from 5 nm to 8 nm

viewing of goligi complex proteins

Can't use fluorescence, proteins too small to see, use electron microscopy, scientists modify antibody by adding gold particle (electron dense material) to be able to see them (called immunogold labelling)

Sorting lysosomal proteins

Mannose-6-phosphate tag on lysosomal proteins, binding to mannose-6-phosphate receptor (MPR) at pH 6.4 (golgi), release at pH 5.5 (endosomes). the longer a vesicle lasts, the more acidic the inside gets

I-cell disease

lysosomal storage disease

Constitutive secretion of vesicles out of cell

default pathway unless signalled to do otherwise, unregulated and continuous secretion, thought to be a default pathway (no tag, no retention in endomembrane system), may not be quite this simple, as short amino acid tags may be implicated in constitutive secretion, glycosylation may play a role in secretion

secretory secretion of vesicles out of cell

signals upon receiving a signal (regulated secretion), secretory vesicles accumulate in the cell, but only fuse with the plasma membrane in response to a specific signal, vesicles bud from TGN and undergo maturation, maturation involves concentrating the proteins, and sometimes also modifying the proteins, immature proteins called zymogen granules

Mast cells

Release histamines - immune system, membrane always left intact - vesicles leave through fusion and release

Exocytosis

Unfavourable - requires the input of energy to move polar head groups through hydrophobic core, membrane stays intact

endocytosis

Important for bringing materials into the cell (membrane transport), play a role in defense (phagocytosis), requires energy when creating vesicle, to pinch bud and move polar head groups through hydrophobic core, requires coat protein

Phagocytosis

Ingestion of large particles (can be more than 0.5 um in diameter), in animals, mainly carried out by neutrophils, macrophages (components of the immune system), mediated by the cytoskeleton, does not require coat proteins

motor proteins

dyneins, myosins, kinesins, walk along cytoskeleton through ATP hydrolysis for each step

dyneins and myosins

walk towards negative end of microtubule

kinesins

walk towards positive end of microtubule

Endolysosomes

Also called clathrin-dependent endocytosis, requires receptors on the outer surface of the plasma membrane, COPI - used for retrograde, COPII - used for antegrade

coat proteins

Energetically favorable, form spherical structures, pulling in membrane, clathrin was the first coat protein identified (Polymerizes into a soccer ball shape), eg. Triskelion - made of 3 large and 3 small polypeptides

clathrin coat assembly

Coat assembly is initiated by ARFs (GTPase) (adaptin binds to target receptor and begins coat assembly, ARF recruits clathrin), clathrin buds, pulling membrane with it (COPI and COPII form in similar manner), lipid bilayer is mediated by dynamin (GTPase) (when it goes from GTP to GDP, it wraps around the stalk and pinches it off). coat is released once the vesicle is formed

Dynamin

PI(4,5)P2 binding domain and GTPase domain, GTP hydrolysis is required for pinching off (energy source)

ARF structure

Has helix with a lipid on it (hydrophobic), allowing it to anchor to cell membrane

For COPI and clathrin at golgi:

initiated by ARF protein

for COPII

utilizes Sar1 for initation

Sar1

GDP - inactive soluble, hydrolysis to GTP - active, has amphipathic helix, can anchor in hydrophobic cell membrane, release from membrane is caused by self-hydrolysis of GTP to GDP

Receptor-mediated endocytosis

ARF or Sar1 embed in membrane and recruit clathrin to form coat, clarthrin can associate with receptors, more than 25 receptors have been identified, a single clathrin-coated pit can probably hold about 1000 receptors, most vesicles enter in the same endosomal compartment

LDL receptor-mediated endocytosis

Lipids have hard time moving in aqueous environment, so hydrophobic nature of them can be hidden, proteins can form a protective layer around lipids, cholesterol largely manufactured in liver, transported in lipoproteins which can hold about 1500 cholesteryl molecules (immature cholesterol) also 800 phospholipids and one protein, released when pH drops, hydrolyzed to cholesterol in lysosomes

pinocytosis

"cell drinking" - pulls in nonspecific items, can pull in LDL

Receptor-independent endocytosis

pinocytosis, vesicles generally about 100 nm in diameter, generally start with clathrin-coated pits or caveolae, caveosomes form from lipid rafts, proteins that enter the cell by caveosomes avoid endosomes and lysosomes, examples of pinocytosis rates: macrophages ingest 3% of their plasma membrane/min; fibroblasts 1% / min, cell volume and surface area remain constant, therefore endocytic and exocytic pathways are equivalent

Rabs

monomeric GTPase family, on vesicle, tethering proteins that stick out of cell membrane, attract Rab to them

SNARE

Soluble NSF Attachment Protein Receptors, are stable, helical bundles of proteins

V-Snare

vesicle SNARE, found on vesicles

T-Snare

Found on plasma membrane (target SNARE)

Vesicle targeting with SNARE

When SNAREs come into contact, they spontaneously wrap together, changing structure, the energy needed for this membrane fusion comes from favourable structural interactions of SNAREs, enzyme and ATP needed to undo and recycle SNAREs after fusion

SNARE in neurons and bacteria toxins

SNARE complex in neurons results in neurotransmitter vesicle fusion and release of neurotransmitter into the synaptic cleft, bacteria-produced toxins: prevents fusion of vesicles, which prevents release of neurotransmitters (Eg: tetanospasmin - tetanus toxin and clostridium botulinum - bacteria that produces toxin used in botox)