BIOL 331 - The Endomembrane System (Lectures 8-12)

endomembrane system

cytoplasmic membrane system, composed of the cytoplasmic membranes (eukaryotic cells), functionally and structurally interrelated group of membranous cytoplasmic organelles (including the endoplasmic reticulum, golgi complex, endosomes, lysosomes, vacuoles)

order of passage of proteins

free ribosomes in the cytoplasm —> rough ER —> golgi complex —> plasma membrane (assuming protein is secreted)

transporting materials between compartments

membrane-bound vesicles shuttle materials between organelles, vesicles bud from donor compartment, transport in a directional manner with motor proteins and the cytoskeleton

vesicles fuse with the membrane of the recipient compartment, cargo released in the destination compartment, membranes fuse so vesicle membrane becomes a part of the recipient compartment’s membrane, escaped resident proteins of the donor compartment can be returned

proteins are directed to the correct destination (eg. secretion, lysosome, membrane), with sorting signals (amino acid sequence or attached oligosaccharides) that are recognized by receptors in the membranes of budding vesicles

biomolecules synthesized in the ER

lipids, cholesterol, steroid hormones, secreted proteins, integral membrane proteins, initial glycosylation of proteins

secretory pathway

transporting materials out of the cell, constitutive or regulated secretion

constitutive secretion

most cells, materials are continually transported in secretory vesicles from their site of synthesis and secreted

contributes to the formation of the plasma membrane

regulated secretion

materials are stored in membrane-bound compartments and only released in response to particular stimuli, eg. endocrine cells that release hormones, pancreatic acinar cells that release digestive enzymes, nerve cells that release neurotransmitters

endocytic pathway

transporting materials into the cell, materials move from the outer surface of the cell to compartments within the cell (endosomes and lysosomes)

endosome

materials are take up and transported to early endosomes for sorting, late endosomes are more acidic than early endosomes, fuse with lysosomes to deliver cargo for degradation

lysosome

hydrolytic (digestive) enzymes and acidic pH, roles in breakdown of material and organelle turnover

autoradiography

following the location of radioactively-labeled materials in a cell, pulse-chase experiments can be used to examine a process that takes place over time

pulse

step 1, radio-labeled amino acids are incorporated in the digestive enzymes being synthesized, exposed to the radio-labelled amino acids for only a very short time

chase

step 2, transfer cells to media with only unlabelled amino acids, enzymes synthesized during this time will not be radio-labeled

endoplasmic reticulum (ER)

a system of membranes and vesicles that encloses the ER lumen (separated from the cytosol), two subcompartments - smooth and rough

rough ER

has ribosomes bound on the cytosolic membrane surface, composed of a network of cisternae, continuous with the outer membrane of the nuclear envelope

extensive in cells with a role in protein secretion, eg. pancreatic acinar cells that secrete hydrolytic enzymes, intestinal cells that secrete mucoproteins, endocrine cells that secrete polypeptide hormones

functions include protein synthesis and initiating addition of sugars

smooth ER

lacks ribosomes, composed of interconnected curved, tubular membranes, continuous with RER

functions include synthesis of steroid hormones (derived from cholesterol), synthesis of membrane lipids, detoxification of organic compounds in the liver, sequestering calcium ions in skeletal and cardiac muscle - role in muscle contraction

free ribosomes

synthesis of 2/3 proteins, ribosomes that are not attached to the ER, proteins are released into the cytosol

synthesis of proteins that remain in the cytosol, peripheral proteins of the cytosolic surface of membranes, proteins transported to the nucleus, mitochondria, and chloroplast

rough ER ribosomes

synthesis of 1/3 proteins, cotranslational translocation (peptides move into the lumen of the ER as it is beind synthesized by the ribosome)

synthesis of secreted proteins, integral membrane proteins and soluble proteins that reside in compartments of the endomembrane system, integral membrane proteins in the plasma membrane

cotranslational translocation (for secreted and soluble proteins)

1. all protein synthesis begins on a free ribosome, signal sequence is at N-terminal end and is 6-15 hydrophobic amino acids

2. signal recognition particle (SRP) binds to the signal sequence and the ribosome, polypeptide synthesis is temporarily halted

3. SRP directs the complex to the ER membrane by interaction with an SRP receptor

4. ribosome/polypeptide are transferred from the SRP to the translocon protein pore in the ER membrane, contact with the signal sequence displaces the translocon’s plug

5. polypeptide enters the ER lumen, upon termination ribosome is released, signal sequence is removed by an enzyme (signal peptidase), protein chaperones (eg. BiP)

cotranslational translocation (for integral membrane proteins)

synthesized using same machinery as secreted proteins, SRP recognizes the hydrophobic transmembrane domain as the signal sequence, transmembrane domains directly enter the lipid bilayer (do not pass through a pore)

1. as polypeptides pass through the translocon, a gate in the pore opens and allows proteins to partition themselves according to their solubility properties, either in the aqueous pore or in the hydrophobic lipid bilayer

2. direction of insertion into the bilayer is dependent on the location of the positively charged amino acids relative to the transmembrane domain

3. cytoplasmic leaflet is more abundant with PS and PI phospholipids (negatively charged), the protein will orient in the membrane such that the positively charged amino acids interact with the relatively negatively charged cytosolic leaflet

positively charged amino acids

arginine (R, Arg), lysine (K, Lys), histidine (H, His)

glycosylation

in the rough ER (and sometimes Golgi complex), majority of proteins produced become glycoproteins, carbohydrate groups have roles as binding sites, aid in proper folding and stabilization, sorting/directing proteins to different cellular compartments, can be N-linked or O-linked

N-linked glycosylation

common, linkage to asparagine (Asn) residues, initiated in the RER

O-linked glycosylation

linkage to serine (Ser) or threonine (Thr) residues, occurs in the golgi complex

N-linked glycosylation in the rough ER

1. first seven sugars are transferred one at a time to a lipid (dolichol pyrophosphate), embedded in the ER membrane, initial assembly is on the cytosolic side, sugars are added by glycosyltransferases

2. dolichol and attached oligosaccharide is flipped across the membrane (cytosolic —> lumen)

3. remaining sugars are attached to dolichol on the cytosolic side, flipped across the membrane and attached to the growing oligosaccharide chain

4. completed oligosaccharide is transferred to an asparagine residue of the polypeptide being translated, transfer by the enzyme oligosaccharyltransferase to an Asn within the sequence Asn-Xxx-Ser/Thr where Xxx is any amino acid except Pro

dolichol pyrophosphate

lipid involved in N-linked glycosylation in the rough ER, embedded in the ER membrane, attaches to sugars

glycosyltransferase

add sugars to lipid during N-linked glycosylation in the rough ER

oligosaccharyltransferase

transfers completed oligosaccharide to an asparagine residue of the polypeptide being translated (Asn with sequence Asn-Xxx-Ser/Thr) where Xxx is any amino acid except Pro

quality control for misfolded proteins

before it can leave the ER…

1. glucosidase I and II remove two glucoses

2. (and 4.2) glycoprotein with one glucose is recognized by calnexin

3. removal of glucose releases protein from chaperone

4. incompletely folded proteins are recognized by UGGT, which detects exposed hydrophobic residues, adds glucose molecule (so it can be detected by calnexin again)

5. properly folded proteins exit

6. improperly folded proteins are degraded in a proteosome in the cytosol

calnexin

a chaperone protein in the ER involved in quality control for misfolded proteins

recognizes glycoproteins with one glucose

UGGT

a conformation sensing enzyme in the rough ER involved in quality control for misfolded proteins, detects exposed hydrophobic residues (of misfolded proteins) and adds a glucose molecule

exiting the ER

1. membrane vesicles with enclosed cargo bud from the ER to travel toward Golgi

2. transport vesicles fuse with one another to form larger vesicles in a region called the ERGIC (endoplasmic reticulum golgi intermediate compartment)

golgi complex

composed of cisternae arranged in a stack, distinct compartments arranged from cis face (closest to ER) to trans face (exit, furthest from the ER)

trans golgi network

furthest from ER, network of tubules and vesicles, sorting station where proteins are segregated into different types of vesicles (heading to the plasma membrane or other)

cis golgi network

closest to ER, interconnected network of tubules, sorting station that distinguishes between proteins that need to be returned to the ER and those that should proceed through the golgi to be sent elsewhere in the cell

protein modification with the golgi complex

newly synthesized proteins leaving the ER are sequentially modified, eg. modification of N-linked carbohydrate chains

• order that sugars are incorporated depends on the location of specific glycosyltransferases (integral membrane proteins in the membrane of the golgi complex)

• glycosylation in the golgi complex can be quite varied

• O-linked carbohydrates are entirely assembled within the golgi

vesicular transport model

golgi cisternae are stable compartments, vesicles carrying cargo bud from one compartment and fuse with the next

evidence: golgi cisternae have different enzymes, lots of vesicles bud from the edges of golgi cisternae

cisternal maturation model

cisternae form at the cis face and move towards the trans face, maturing as they move

evidence: drugs blocking vesicle formation at the ER leads to the golgi complex disappearing, certain large materials, eg. collage, move from cis to trans without ever appearing in smaller vesicles (always a large compartment)

***overall a stronger model

anterograde transport

from the cis face to the trans face of the golgi (forward)

retrograde transport

from the trans face to the cis face of the golgi (backward), resident golgi and ER enzymes

types of coated vesicles

COPI, COPII, clathrin-coated

COPII-coated vesicles

move cargo forward (anterograde), ER to Golgi

select and concentrate certain proteins for transport in vesicles by interacting with transmembrane proteins that have ER export signals

enzymes destined for the Golgi complex (eg. glycosyltransferases), proteins involved in vesicle docking and fusion, protein receptors that bind soluble cargo

Sar1

a COPII coat G protein (binds GDP)

guanine exchange factor (GEF)

recruits Sar1-GDP and replaces GDP with GTP

Sar1-GTP

undergoes conformational change so that it inserts into the cytoplasmic leaflet and bends the membrane

Sec24

the primary adaptor protein that interacts with membrane proteins (forms a dimer with Sec23 to further bend the membrane)

Sec13/Sec31 forms an outer structural cage around the membrane

disassembly is triggered by hydrolysis of GTP bound to Sar1

COPI-coated vesicles

coat is made up of a protein complex called coatamer, which forms a thick protein coat directly on the membrane, Arf1 involved

retrograde transport of proteins from golgi to ER (eg, golgi resident enzymes, ER resident proteins)

proteins that reside in the ER contain a retrieval signal (different for soluble ER proteins and membrane ER proteins), each compartment in the endomembrane system may have its own retrieval signal

Arf1

a membrane-bending G protein, GTP form bends membrane, involved in retrograde transport of proteins with COPI-coated vesicles

retrieval signal for soluble ER proteins

usually contain the signal lys-asp-glu-leu (KDEL), recognized by a KDEL receptor (shuttle between cis Golgi and ER compartments)

retrieval signal for membrane ER proteins

usually lys-lys-X-X (KKXX)

vesicle fusion

1. movement of the vesicle towards the specific target compartment, mediated by microtubules and motor proteins

2. tethering vesicles to the target compartment - tethering proteins (rod-shaped/fibrous and multiprotein complex), G proteins (Rabs)

3. docking vesicles to the target compartment - SNARE proteins form complexes with another SNARE protein (v-SNARE, t-SNARE)

4. fusion between vesicle and target membrane - interactions between t- and v-SNARES pull lipid bilayers together with enough force to cause fusion

two types of tethering proteins

rod-shaped/fibrous (longer)

multiprotein complex (closer)

involved in vesicle fusion

Rabs

G proteins (60+ in humans) help to determine specificity, recruit specific tethering proteins, and interact with motor proteins

SNARE proteins

integral membrane proteins, 35+ different proteins in specific compartments, eg. v-SNAREs and t-SNAREs involved in vesicle fusion

v-SNARE

put into transport vesicles during budding (for vesicle fusion), forms 4 stranded bundles with other SNARE protein

t-SNARE

located in the target membrane (for vesicle fusion), forms 4 stranded bundles with other SNARE protein

determines the ability of a vesicle to fuse to a specific membrane

the specific combination of Rabs, SNARES, and tethering proteins

lysosomes

contain at least 50 hydrolytic enzymes (with an acidic optimal pH), pH is ~4.6 (maintained by a proton pump)

roles of lysosomes

1. breakdown of material brought into the cell by endocytosis, eg. phagocytic cells in mammals

2. organelle turnover (autophagy) - regulated destruction and replacement of cell organelles

organelle turnover (autophagy)

organelle is surrounded by a double membrane structure (autophagosome) - inner and outer autophagosomal membranes

autophagosome fuses with lysosome —> autolysosome

starved cells exhibit increased autophagy

inner autophagosomal membrane

cargo sequestration

outer autophagosomal membrane

fusion with the lysosomal membrane

clathrin-coated vesicles

move materials from the trans golgi network to endosomes, lysosomes, plant vacuoles, for endocytosis, etc.

composed of clathrin, GGA adaptor, mannose 6-phosphate receptor (MPR)

targeting lysosomal enzymes to lysosomes

• soluble lysosomal enzymes are recognized by enzymes that add phosphate groups to mannose sugars of N-linked carbohydrate chains

• the phosphorylated mannose (mannose 6-phosphate) residues act as a sorting signal, directing proteins to the lysosome

1. mannose residues are phosphorylated in the Golgi (mannose 6-phosphate)

2. lysosomal enzymes are incorporated into a clathrin-coated vesicle at the TGN

3. MPRs separate from the lysosomal enzymes and are returned to the Golgi

4. clathrin coat is disassembled and lysosomal enzymes are delivered to a sorting endosome and onto a lysosome

clathrin

coat protein that forms structural scaffold on vesicles

GGA adaptor

connects clathrin to MPRs, helps gather up lysosomal enzymes, (clathrin coated vesicles)

has multiple domains - binds Arf1-GTP, clathrin, and cytosolic tails of the MPRs (adaptor = physically links two or more components)

results in concentrating lysosomal enzymes into vesicles

mannose 6-phosphate receptor

transmembrane protein that recognizes and captures proteins with the mannose 6-phosphate signal

Arf1-GTP

G protein that binds to the membrane and initiates formation of the budding vesicle and binding of the other coat proteins, induces membrane curvature when bound together

transport of secreted proteins

golgi cisternae move continually toward the TGN which fragments into vesicles and tubules, constitutive secretion may be the ‘default’

endocytosis

two types - bulk-phase and receptor-mediated

bulk-phase endocytosis

aka pinocytosis, non-specific uptake of extracellular fluids, and any molecules that happen to be present

receptor-mediated endocytosis

clathrin-mediated, specific molecules binding to receptors on the extracellular surface of the plasma membrane, selective uptake, eg. hormones, growth factors, certain nutrients

AP2 complex (adaptor)

links cytoplasmic tails of plasma membrane receptors with clathrin

triskelion

a clathrin molecule is composed of three heavy chains and three light chains

dynamin

a G protein required for the clathrin-coated vesicle to bud from the membrane, subunits polymerize to form a ring, GTP hydrolysis induces a movement in the ring, vesicle is cleaved and the ring disassembles

recycling pathway

one outcome of endocytosis, housekeeping receptors mediate uptake of materials that will be used by the cell (cholesterol iron, etc)

receptors are first transported to an early endosome for sorting

ligands dissociate due to acidic pH

receptors are concentrated into a recycling compartment of the early endosome

vesicles return receptors to the cell surface to be used again

degradation pathway

one outcome of endocytosis, signalling receptors bind ligands that affect cellular activities (hormones, growth factor)

first transported to the early endosome for sorting, early endosome matures into late endosome

late endosome fuses with lysosome for receptor degradation

receptor degradation prevents the cell from being further stimulated by the hormone/growth factor - reduces sensitivity to a molecule