Lec 14 - Functions of the ER

What are the two morphologically distinct regions of the endoplasmic reticulum

Rough ER (ribosome-studded) and smooth ER (no ribosomes), which are contiguous parts of one organelle

Are ER and nuclear membranes contiguous (touching with no gaps), and are they considered separate

They are contiguous but considered separate due to distinct functions

Approximately what fraction of total cell volume is rough ER

About ~10% of total cell volume

Approximately what fraction of total cellular membrane is ER

About ~30–50% of total cellular membrane is ER

What type of membrane structure does the ER have

A single membrane bilayer distributed throughout the cytoplasm

What ion is sequestered and stored in the ER lumen

Ca2+, which can be released in response to action potentials or second messenger signaling

Which ER region has ribosomes and what side of the membrane are they on

Ribosomes stud the rough ER and bind only the cytosolic leaflet of the ER membrane

What does smooth ER look like compared to rough ER by EM

Smooth ER appears as ribosome-free tubular/globular cisternae, while rough ER is ribosome-studded

What is the major biosynthetic role of the rough ER

Synthesis and processing of secreted proteins, membrane proteins, and lysosomal enzymes

What are major functions of the smooth ER

Membrane lipid synthesis, steroidogenesis, detoxification, triacylglycerol and cholesterol ester synthesis, and Ca2+ storage

How were rough vs smooth ER functions distinguished experimentally

By subcellular fractionation on sucrose gradients into light (smooth) and heavy (rough) microsomes followed by functional assays

Which microsomes are low density and float at low sucrose

Smooth microsomes

Which microsomes are high density and float at high sucrose

Rough microsomes

What happens when cytosol + mRNA for a cytosolic protein are mixed with energy and tRNAs in vitro

Protein is produced without ER membranes

What was required to produce secreted/membrane proteins in vitro translation assays

Addition of rough microsomes to the reaction

What targets a translating ribosome–nascent chain complex to the ER

An N-terminal signal sequence that mediates co-translational targeting to the ER

Roughly what percentage of cellular proteins are secreted or membrane proteins requiring ER targeting

About ~30%

List membranes whose proteins are synthesized in the RER

Plasma, ER, lysosome, nuclear, and peroxisome membranes

Which soluble proteins are synthesized in the RER

Secreted proteins and luminal proteins of organelles like lysosomal enzymes

Where are enzymes located that add carbohydrate groups to newly synthesized proteins and lipids

In the ER lumen for initial glycosylation steps described in this session

On which leaflet are phospholipid synthesis enzymes located in smooth ER

The cytosolic leaflet of the smooth ER membrane

What are the key early steps to generate phosphatidic acid at the ER

Glycerol-3-phosphate + fatty acyl-CoAs via GPAT and LPAAT form phosphatidic acid

What converts phosphatidic acid to diacylglycerol (DAG)

A phosphatase removes the phosphate to yield DAG

What enzyme class adds head groups to DAG to make phospholipids

Phosphotransferases add head groups such as choline, ethanolamine, serine, or inositol

What is the key intermediate common to phospholipid synthesis

Diacylglycerol (DAG)

Which enzyme makes phosphatidylserine in ER and where is PS decarboxylated to PE

Phosphatidylserine synthase acts in ER; phosphatidylserine decarboxylase in mitochondria converts PS to PE

Why can newly synthesized lipids not spontaneously cross the bilayer

Polar headgroups are hydrophilic and require proteins to traverse the bilayer

Which proteins move lipids from cytosolic to luminal leaflet in ER

Floppases move lipids to the luminal leaflet to balance bilayer growth

What do flippases, floppases, and scramblases do

Flippases move specific lipids to cytosolic leaflet, floppases to luminal/exoplasmic leaflet, and scramblases equilibrate lipids bidirectionally

Which leaflet initially receives newly synthesized phospholipids at ER

The cytosolic half of the bilayer

Where is ceramide synthesized and from what substrates

In the smooth ER from palmitate (palmitoyl-CoA), serine, and a fatty acyl-CoA

Name a key intermediate in ceramide synthesis highlighted in lecture Dihydrosphingosine (sphinganine)

Where are sphingomyelin and glycolipids synthesized from ceramide

In the Golgi apparatus via addition of phosphorylcholine (for sphingomyelin) or sugars (for glycolipids)

Where are TAGs synthesized in cells

In the smooth ER via de novo synthesis or re-esterification of dietary monoacylglycerols

Which enzymes sequentially produce TAG from glycerol-3-phosphate in de novo synthesis

GPAT → LPAAT → phosphatidate phosphatase → DGAT

Can adipocytes use glycerol directly to make TAGs, and why or why not

No, adipocytes lack glycerol kinase and rely on DHAP from glucose to make glycerol-3-phosphate

Where do apoproteins meet TAGs to form chylomicrons, and where are chylomicrons assembled

Lipids meet apoproteins in transitional ER; chylomicrons are assembled in the Golgi and secreted to lymph then blood

Where do early steps of cholesterol synthesis occur and where is the terminal site

Early steps occur in cytoplasm; the ER is the terminal site where lanosterol is converted to cholesterol

What is the rate-limiting enzyme of cholesterol synthesis and where is it located

HMG-CoA reductase, an integral ER membrane protein

How many HMG-CoA reductases were noted and what do they produce

Two were noted: a mitochondrial form producing ketones and an ER membrane form producing cholesterol

Which cholesterol synthesis intermediates serve in protein prenylation (lipidation)

Farnesyl-PP and geranyl-PP

Where are most steroid hormone synthesis enzymes located

In the smooth ER, with some steps in mitochondria depending on the hormone

Which steroid hormone class has final steps in mitochondria per the slides

Mineralocorticoids

What hormone triggers cortisol synthesis and release from the adrenal gland

ACTH (adrenocorticotropic hormone)

Outline the cortisol synthesis shuttle between organelles Cholesterol ester → cholesterol → mitochondria (cholesterol → pregnenolone) → ER (progesterone → 11-deoxycortisol) → mitochondria (cortisol)

How do ER and mitochondria coordinate during steroidogenesis

Multiple sequential steps are partitioned between the two compartments for regulated synthesis and shuttling of intermediates

What are cytochrome P450 enzymes and where do they act for detoxification

ER-resident oxidoreductases mediating Phase I reactions to increase solubility of xenobiotics

What completes detoxification after Phase I and what reactions occur

Phase II transferases (e.g., sulfation, glucuronidation, glutathione conjugation) produce more water-soluble metabolites

What cofactors deliver electrons to P450s for O2 activation

NADPH donates electrons via cytochrome P450 reductase containing FAD and FMN

What is MEOS and when is it engaged

Microsomal ethanol-oxidizing system in smooth ER activated by excess ethanol or other compounds requiring solubilization

Which P450 isoform oxidizes ethanol in MEOS

CYP2E1

What cofactor does MEOS use and what are the products of ethanol oxidation

Uses NADPH and O2 to produce acetaldehyde and H2O

How does CYP2E1’s Km for ethanol compare to ADH1 family members

CYP2E1 has much higher Km (≈11 mM) than ADH1 (≈0.02–5 mM), contributing more at high ethanol levels

What clinical effect does chronic ethanol have on CYP2E1 levels

Induces CYP2E1 ~5–10-fold and other P450s 2–4-fold, increasing ethanol clearance and potential toxicity

What are risks of CYP2E1 induction beyond tolerance

Excess acetaldehyde and free radical generation increase hepatic injury and cirrhosis risk

What is the approximate blood ethanol concentration for legal intoxication listed in the slides

About 0.08% (≈18 mM)

How can ethanol interact with phenobarbital metabolism

Ethanol inhibits the phenobarbital-oxidizing P450 system, risking toxic barbiturate accumulation when co-consumed

Which toxic intermediate is formed from acetaminophen by P450s, and which isoform is implicated

NAPQI, produced by CYP2E1

Under normal dosing, what proportion of acetaminophen forms NAPQI and how is it detoxified

Less than 10% forms NAPQI, which is detoxified by glutathione conjugation

Why are chronic alcohol users at higher risk of acetaminophen toxicity

Induced CYP2E1 channels more acetaminophen to NAPQI, overwhelming glutathione and causing hepatocyte injury

What is an effective treatment for acetaminophen poisoning and why

N-acetylcysteine replenishes cysteine for glutathione synthesis, enhancing detoxification of NAPQI

Why should patients on statins avoid grapefruit per the slides

Grapefruit inhibits CYP3A4, reducing statin metabolism and causing accumulation with muscle toxicity

Where is glucose-6-phosphatase (G6Pase) located and where is its active site

In the ER membrane with its active site facing the ER lumen

Which ER transporters participate in G6P handling and in which directions

T1 transports G6P into ER lumen; T2 and T3 return glucose and Pi to cytosol

Why are glycogen granules juxtaposed to ER

The final dephosphorylation step of glycogenolysis requires ER-localized G6Pase, favoring proximity

What are peroxins (PEX) and what do they do

Peroxins are proteins required for peroxisome biogenesis and matrix enzyme import

Which peroxins are structural membrane proteins and how do they reach peroxisomes

PEX13, PEX14, and PEX17 are structural; they are made in the ER and reach peroxisomes via fusion of ER-derived vesicles

Which peroxins function as soluble cytosolic receptors for peroxisomal cargo in this lecture

PEX5 and PEX7

What targeting sequence does PEX5 recognize

The serine–lysine–leucine (SKL) tripeptide (PTS1)

How do PEX5/PEX7 receptor functions differ from nuclear import receptors

They deliver cargo to the peroxisome membrane and drop it off without entering the organelle

Does PEX7 recognize the same sequence as PEX5

No, PEX7 recognizes a different peroxisomal targeting sequence than SKL

Which phospholipid headgroups can be added to DAG in ER to form major membrane lipids

Choline (PC), ethanolamine (PE), serine (PS via PS synthase), and inositol (PI)

Where are glycolipids primarily synthesized

In the rough ER and Golgi, with final glycosylation steps in the Golgi

Where does phosphatidylserine decarboxylation occur to produce PE

In mitochondria

What processes enable intestinal absorption of dietary fats for TAG resynthesis

Emulsification by bile salts and hydrolysis by pancreatic lipases to monoacylglycerol and fatty acids for re-esterification in SER

After resynthesis in enterocytes, how are lipids transported systemically

Packaged into chylomicrons with apoproteins and secreted to lymph then blood

What is the topological equivalence of organelle lumen relative to the cell

Organelle lumen is equivalent to the extracellular space in terms of membrane orientation

What side of any organelle membrane corresponds to the cytosolic face

The side facing the cytosol maintains cytosolic identity across organelles

Define co-translational transport in one line Simultaneous translation and ER translocation of nascent polypeptides guided by an ER signal sequence

Name the headgroup donor commonly used to add choline during PC synthesis CDP-choline

Which phospholipid synthesis enzymes were highlighted by name

GPAT and LPAAT (acyltransferases)

What central principle dictates organelle-specific reactions emphasized in the lecture

Reactions occur only where all required components co-localize, dictating organelle-specific pathways

What is stored as cholesterol esters before steroidogenesis activation

Cholesterol is stored as cholesterol esters and mobilized upon ACTH signaling

What distinguishes Phase I vs Phase II detoxification in terms of solubility

Phase I introduces polar groups; Phase II conjugates to markedly increase water solubility for excretion

Does the ER contain glucose transporters relevant to G6P hydrolysis

Yes—T1 (G6P import) and T2/T3 (glucose and Pi export) in the ER membrane

What organelle site is emphasized as terminal for de novo cholesterol synthesis

The ER, where lanosterol is converted to cholesterol

Which organelles collaborate for mineralocorticoid final synthesis steps per slides

ER and mitochondria, with final step in mitochondria

What is the function of cytochrome P450 reductase in the detox scheme

Transfers electrons from NADPH to P450 to activate O2 for oxidative reactions

Why can excessive ethanol plus acetaminophen be dangerous

Ethanol induces CYP2E1, increasing NAPQI from acetaminophen beyond glutathione capacity, causing hepatotoxicity

In what cellular fraction are MEOS enzymes historically found upon cell disruption

Microsomes, the ER-derived membrane fraction after centrifugation

What is the role of floppases in maintaining bilayer growth symmetry

They move newly made lipids to the luminal leaflet so both halves expand equally

Which lipid class’s synthesis begins in ER but finishes in Golgi with sugar additions

Glycosphingolipids synthesized from ceramide

What is the biochemical reason adipocytes require glucose to store fatty acids

Lack of glycerol kinase necessitates DHAP from glycolysis to generate glycerol-3-phosphate

Which organelle is explicitly noted as not part of the endomembrane system in this lecture

Mitochondria, which require distinct import mechanisms for proteins

What headgroup addition converts ceramide to sphingomyelin and where

Addition of phosphorylcholine in the Golgi

Does PEX5 enter the peroxisome with cargo according to the lecture

No, it delivers and releases cargo at the membrane without entering

What shared concept ties ER lumen topology to cellular architecture

Organelle lumens mirror extracellular orientation for membrane proteins and glycans