Cell Bio Endoplasmic Reticulum and Golgi Apparatus

ETC complex IV more info

- complex IV pumps 4 H+ per O2 reduced but requires 2 electron carriers (4 total e-s)

- per NADH, 2 H+ are pumped via complex IV

endoplasmic reticulum (ER)

- largest continuous membrane network in the cell

- continuous with the nuclear envelope

- central hub for protein and lipid biosynthesis

- architecture adapts to cellular demand

ER structure

- cisternae

- tubules

- form reflects function; balance shifts with cell needs

- continuous internal lumen throughout the network

cisternae

- flattened sacs

- site of protein synthesis near the nucleus

tubules

- branching network

- flexible for lipid metabolism and transport

rough ER (RER)

- ribosome studded

- entry for secretory/membrane proteins

- sheet like

smooth ER (SER)

- ribosome free

- major site for lipid related processes

- smooth tubular membranes

rough and smooth ER

- regions are continuous and interconvertible

- division of labor enhances efficiency

transmission electron microscopy of RER and SER

- TEM reveals structural differences

- arrangement reflects functional specialization

distribution of ER in different subtypes

- ER abundance/ratio varies by tissue demand

- ER plasticity supports specialization

secretory cells ER

expanded RER (eg exocrine pancreas)

membrane/lipid-active cells ER

expanded SER (eg liver, steroidogenic tissues)

functions of RER

- co-translational import of proteins

- folding and quality control

- initial N-linked glycosylation

- gateway to secretory pathway

co-translational import overview

- signal peptide directs ribosome to ER

- signal recognition particle (SRP) binds sequence

- SRP-ribosome complex docks at ER membrane (Translocon)

- translocon enables entry into ER lumen during polypeptide synthesis

signal peptide

short N-terminal motif that marks ER entry

tripartite features of signal peptide recognition

- positively charged N-region

- hydrophobic core

- cleavage region

signal peptide recognition

- tripartite features

- recognition occurs as the peptide emerges from the ribosome

- necessary and sufficient: swapping in a signal peptide re-routes proteins to ER

signal recognition particle (SRP)

- SRP = ribonucleoprotein complex

-> Alu domain and S domain

- binds hydrophobic signal peptide during translation

- temporarily pauses elongation

- directs ribosome-nascent chain to ER membrane

SRP receptor and translocon mechanism

- SRP-ribosome complex binds SRP receptor (ER membrane)

- ribosome aligns with Sec61 translocon

- GTP hydrolysis triggers SRP release

- translation resumes; chain threaded into ER

ribosome docking and translocation

- ribosome sits tightly over Sec61 translocon channel

- translation resumes into ER lumen seamlessly

- signal peptide usually cleaved by signal peptidase

- chaperones assist folding as chain enters lumen

BiP

ER-resistant Hsp70 chaperone

chaperones (BiP) in folding

- binds nascent chains entering lumen

- prevents misfolding and aggregation

- uses ATP hydrolysis to stabilize folding intermediates

disulfide bond formation (disulfide isomerase)

- favored in oxidizing ER lumen

- stabilizes tertiary and quaternary structures

- bonds can be rearranged to correct mispairing (isomerization)

- catalyzed by protein disulfide isomerase (PDI)

N-glycosylation initiation in ER

- addition of oligosaccharides to asparagine residues

- occurs co-translationally in the ER lumen

- catalyzed by oligosaccharyl transferase (OST)

- essential for folding, stability, and quality control

quality control: misfolded proteins retained

- only properly folded proteins exit the ER

- misfolded proteins retained by chaperones

- calnexin/calreticulin cycle monitors glycoprotein folding

- persistent misfolding -> ER-associated degradation (ERAD)

ER-associated degradation (ERAD)

- misfolded proteins retrotranslocated to cytosol

- tagged with ubiquitin for proteasomal degradation

- prevents accumulation of defective proteins in ER

- protects cell from proteotoxic stress

unfolded protein response (UPR)

- triggered by ER stress: accumulation of misfolded proteins

- activates three main sensors (IRE1, PERK, ATF6)

- expands folding capacity, reduces translation

- prolonged stress -> apoptosis

cystic fibrosis and misfolded CFTR

- cystic fibrosis caused by mutations in CFTR chloride channel

- leads to defective chloride transport in epithelia

- ΔF508 mutation -> misfolding in ER

- misfolded CFTR retained and degraded via ERAD

functions of the SER

- lipid and steroid biosynthesis

- detoxification of drugs/toxins

- calcium storage and release

- specialized roles in different tissues

lipid biosynthesis

- cytosolic leaflet of smooth ER

- phospholipids, cholesterol, and steroid hormones produced

- asymmetry: lipids added mainly to cytosolic leaflet

- scramblases and flippases redistribute lipids

cytosolic leaflet of smooth ER

primary site of lipid synthesis

cholesterol and steroid hormone production

- SER synthesizes cholesterol; cells also import it (LDL)

- StAR moves cholesterol into mitochondria on demand

- cholesterol first committed step to pregnenolone in mitochondria (CYP11A1)

- steroidogenesis proceeds in adrenal cortex and gonads (tissue-specific enzymes)

SER synthesizes cholesterol; cells also import it (LDL)

Acetyl-CoA -> mevalonate -> isoprenoids -> squalene -> cholesterol

detoxification (cytochrome P450 in SER)

- SER-localized CYP450s oxidize drugs/xenobiotics

- require heme + NADPH-CYP450 reductase (+/- cytochrome b5)

- induction/inhibition and polymorphisms alter drug levels

detoxification (cytochrome P450 in SER): reaction

RH + O2 + NADPH -> ROH + H2O

calcium storage/release (sarcoplasmic reticulum)

- SERCA pumps load Ca2+

- calsequestrin buffers luminal Ca2+

- RyR channels release Ca2+ on excitation

- rapid reuptake (SERCA) ends contraction

- phospholamban modulates cardiac SERCA

sarcoplasmic reticulum (SR)

specialized smooth ER wrapped around myofibrils (actin, myosin)

liver (hepatocyte) cell specific adaptations

- expanded SER for xenobiotic metabolism

- RER for secreted proteins (eg albumin)

adrenal cortex cell specific adaptations

abundant SER + mitochondria for steroidogenesis (cholesterol -> hormones)

cell specific adaptations

- distinct enzyme complements and organelle architecture reflect tissue demands

- dynamic remodeling

retrotranslocon

opposite of translocons, go opposite direction (out)

dynamic remodeling

hormones/drugs can upregulate pathways and expand ER

golgi structure

polar stack of flattened cisternae: cis -> medial -> trans

cis cisternae in golgi

- entry

- adjacent to ER

- receives COPII vesicles

medial cisternae in golgi

intermediate processing compartment

trans/TGN cisternae in golgi

- exit

- late processing + staging for outbound traffic

cisternal maturation model

- cisternae themselves mature: cis -> medial -> trans

- cargo stays within the same cisterna as it matures

- golgi enzymes move backward (retrograde) to re-establish compartment identity

- explains transport of large cargo (e.g. procollagen)

vesicular transport model

- cisternae are static

- cargo moves forward in vesicles

- COPII

- COPI

- small/medium cargo packaged into forward carriers

- retrograde COPI retrieves ER residents and golgi enzymes

COPII in vesicular transport model

ER -> cis-golgi

COPI in vesicular transport model

- anterograde here

- cis -> medial -> trans

evidence for cisternal maturation model

- large cargo (procollagen) stays within a maturing cisterna

- cis/medial/trans markers exchange over time

evidence for vesicular transport model

- abundant COPI/COPII buds

- cargo receptors enrich small/medium cargo

- rapid pulse-chase transfer between cisternae

evidence for hybrid model (cisternal maturation and vesicular transport)

cells likely use both depending on cargo size/class and demand

glycosylation modifications in golgi

- N-linked processing

- O-linked initiation

- proteoglycans

- compartmentalization

glycosylation modifications in golgi: compartmentalization

enzyme sets are zoned (cis -> medial -> trans) to produce ordered, stepwise modifications

glycosylation modifications in golgi: N-linked processing

- ER core

- golgi (cis->medial)

glycosylation modifications in golgi: N-linked processing: ER core

- Glc3Man9GlcNAc2 -> trimmed

- quality check completed in ER

glycosylation modifications in golgi: N-linked processing: golgi (cis -> medial)

- mannose trimming

- (medial -> trans): add GlcNAc, Gal, Fuc, Sia

glycosylation modifications in golgi: N-linked processing: end types

- high-mannose

- hybrid

- complex

glycosylation modifications in golgi: N-linked processing: functions

- stability

- trafficking signals

- cell-cell interactions

glycosylation modifications in golgi: O-linked initiation

- initiates golgi on Ser/Thr

- GalNAc-T adds first GalNAc -> chains extend (Gal, GlcNAc)

- prominent in mucins

- affects viscosity, protection, signaling

glycosylation modifications in golgi: proteoglycans

- linker on Ser

- GAG types

- roles

glycosylation modifications in golgi: proteoglycans: linker on Ser

Xyl-Gal-Gal-GlcA -> tehn glycosaminooglycan (GAG) chain polymerized

glycosylation modifications in golgi: proteoglycans: GAG types

- chondroitin/dermatan

- keratan (tissue specific)

glycosylation modifications in golgi: proteoglycans: roles

- ECM structure

- growth factor binding

- filtration

- signaling

mannose trimming/complex sugar addition

- cis/medial golgi

- medial/trans golgi

- branching increases

- glycan remodeling tunes stability, recognition, and half life

mannose trimming/complex sugar addition: cis/medial golgi

mannosidases trim ER core (Man9 -> Man5)

mannose trimming/complex sugar addition: medial/trans golgi

- add GlcNAc -> Gal -> Sia (+/- Fuc)

- build hybrid/complex N-glycans

mannose trimming/complex sugar addition: branching increases

(bi-, tri-, tetra-, antennary) with sequential GlcNAc transferases

congenital disorders of glycosylation

- inborn errors of protein glycosylation -> multisystem disease

- Type I vs Type 2

common features of congenital disorders of glycosylation

- neurologic

- liver/coagulation

- GI/endocrine

congenital disorders of glycosylation: Type 1

- assembly/transfer

- ER

congenital disorders of glycosylation: Type 2

- processing

- golgi

sorting pathways

- trans-golgi network (TGN)

- secretion

- plasma membrane

- lysosomes

trans-golgi network (TGN)

sorting hub: cargo tagged/packaged for destinations

secretion

constitutive vs regulated

constitutive secretion

- default export

- continuous export from TGN -> surface/ECM

- ex: albumin, membrane lipids

regulated secretion

- stored

- stimulus triggered exocytosis

- cargo condenses into granules

- ex: insulin, neurotransmitters, digestive enzymes

plasma membrane

delivery of integral proteins/lipids to specific domains

lysosomes

M6P-tagged hydrolases sorted to degradative pathway

sorting at TGN

bulk flow vs signal/receptor-medicated packaging

endomembrane system: ER-golgi trafficking

- ER -> golgi via COPII carriers from ER exit sites

- cis-medial-trans golgi processing -> TGN sorting hub

- retrograde COPI returns ER/golgi residents and receptors

- net flow

endomembrane system: ER-golgi trafficking: net flow

- synthesize in ER

- refine in golgi

- route at TGN

protein flow

ER -> golgi -> final destination

ER step of protein flow

synthesis and folding/QA -> COPII export from ER exit sites

golgi step of protein flow

(cis -> medial -> trans)

- sequential trimming/adding of sugars -> TGN hub

TGN sorting step of protein flow

- to constitutive secretion

- regulated granules

- plasma membrane

- lysosomes (M6P)

retrieval signals

KDEL

KDEL

- KDEL C-terminal motif marks ER-resident soluble proteins

- KDEL receptor in cis-golgi binds escaped proteins (pH dependent)

- COPI vesicles return them to ER; release in ER conditions

- maintains ER protein composition despite bulk flow