Membranes
Cell Membrane: Functions & Structure
Q: What are the main functions of the cell membrane?
A: Protection, selective permeability, communication, and structural support.
Q: What is the structural organization of the cell membrane?
A: A phospholipid bilayer with embedded proteins, cholesterol, and carbohydrates.
Lipid Bilayer: Composition & Structure
Q: What are the three main types of lipids in the membrane?
A: Phospholipids, cholesterol, and glycolipids.
Q: What is the function of cholesterol in the membrane?
A: Maintains membrane stability and fluidity.
Q: What is the role of glycolipids?
A: Involved in cell recognition and communication.
Membrane Proteins
Q: What are the three types of membrane proteins?
A: Peripheral, integral, and lipid-linked proteins.
Q: How do integral proteins interact with the membrane?
A: They span the membrane, with portions exposed on both sides.
Q: What are the functions of membrane proteins?
A: Structural support, cell signaling, transport, and cell-cell communication.
Nucleus
Q: What is the structure of the nuclear membrane?
A: Two lipid bilayers with a perinuclear space between them.
Q: What is the function of the nucleus?
A: Protects genetic material and controls molecule exchange via nuclear pores.
Endoplasmic Reticulum (ER)
Q: What are the functions of the ER?
A: Protein and lipid synthesis, inter-organelle communication, and organelle division.
Golgi Apparatus
Q: What is the function of the Golgi apparatus?
A: Modifies, sorts, and packages proteins for transport.
Q: What is the Trans-Golgi Network (TGN)?
A: The final sorting station where proteins are packed into vesicles.
Vesicular Transport
Q: What are vesicles used for?
A: Transporting molecules via exocytosis, endocytosis, and intracellular transport.
Mitochondrion
Q: What are the main compartments of the mitochondria?
A: Outer membrane, inner membrane (with cristae), and the matrix.
Q: What is the function of the mitochondrial intermembrane space?
A: Plays a key role in oxidative phosphorylation.
Phospholipids
Q: What are the two main types of phospholipid fatty acid chains?
A: Saturated (no double bonds) and unsaturated (contains double bonds).
Q: What is the structure of a phospholipid?
A: A hydrophilic phosphate head and two hydrophobic fatty acid tails.
Types of Phospholipids
Q: What is the most abundant phospholipid in cell membranes?
A: Phosphatidylcholine (PC).
Q: What is the function of phosphatidylserine (PS)?
A: Found on the inner membrane; signals apoptosis when exposed on the outer surface.
Sphingolipids
Q: What is the main function of sphingolipids?
A: Structural integrity and cell signaling.
Q: Where is sphingomyelin commonly found?
A: In the myelin sheath of neurons.
Cholesterol & Membrane Fluidity
Q: How does cholesterol influence membrane fluidity?
A: Prevents stiffness at low temperatures and excessive fluidity at high temperatures.
Q: What happens to the bilayer at very high and low temperatures?
A: High temp: too fluid, loses barrier function. Low temp: rigid, gel-like state.
Glycolipids & Glycoproteins
Q: What is the role of glycolipids in the membrane?
A: Cellular recognition and stability.
Q: What is the function of glycoproteins?
A: Facilitate cell-cell communication and transport.
Lipid Rafts & Flipases
Q: What are lipid rafts?
A: Membrane microdomains rich in cholesterol and sphingolipids, involved in signaling.
Q: What is the role of flipases?
A: Enzymes that help maintain lipid asymmetry by flipping phospholipids across the bilayer.
Energy Storage & Lipid Signaling
Q: How are triglycerides stored?
A: In adipose tissue, where they undergo continuous synthesis and breakdown.
Q: What is the role of diacylglycerol (DAG) and phosphatidylinositol phosphates (PIPs)?
A: Involved in calcium-mediated activation of protein kinase C.
Signal Writers, Readers, Erasers
Q: What are signal writers, readers, and erasers?
A: Writers add modifications (e.g., kinases). Readers recognize modifications (e.g., binding phosphorylated sequences). Erasers remove modifications (e.g., phosphatases).
Unsaturated Fatty Acids
Q: How do unsaturated fatty acids differ from saturated ones?
A: They have C=C bonds, causing kinks that prevent tight packing, lower melting points, and make them liquid at room temp.
Acetyl-CoA & Fatty Acid Synthesis
Q: How is acetyl-CoA formed?
A: Pyruvate is converted to acetyl-CoA, producing NADH and releasing CO₂.
Q: What enzyme catalyzes the first step of fatty acid synthesis?
A: Acetyl-CoA Carboxylase (ACC) converts acetyl-CoA → malonyl-CoA, requiring ATP.
Q: What is the key enzyme in fatty acid synthesis?
A: Fatty Acid Synthase (FAS) catalyzes the elongation of fatty acids using acetyl-CoA and NADPH.
Palmitate Synthesis
Q: When does the fatty acid chain exit synthesis?
A: At 16 carbons, forming palmitate (16:0), which can be elongated to stearate (18:0).
Unsaturated Fatty Acid Synthesis
Q: What enzyme creates double bonds in fatty acids?
A: Desaturases, e.g., stearoyl-CoA desaturase, which converts stearoyl-CoA → oleoyl-CoA.
Membrane Synthesis
Q: Where are phospholipids synthesized?
A: On the cytosolic face of the smooth ER (SER) by membrane-associated enzymes.
Q: How do organelles acquire lipids?
A: Via vesicular transport, membrane contact sites, and lipid transfer proteins.
Cholesterol Synthesis
Q: What is the rate-limiting step?
A: HMG-CoA Reductase (HMGCR) converts HMG-CoA → mevalonate.
Q: Where does cholesterol synthesis occur?
A: In the cytoplasm & ER from acetyl-CoA.
Q: What are the steps in cholesterol synthesis?
Acetyl-CoA → HMG-CoA
HMG-CoA → Mevalonate (HMGCR)
Mevalonate → Isoprenoids
Isoprenoids → Squalene
Squalene → Cholesterol
Q: How is cholesterol synthesis regulated?
A: HMGCR is inhibited by cholesterol & statins.
Q: What role does cholesterol play in membranes?
A: Maintains fluidity, stability, and raft formation by interacting with phospholipids.
Cardiolipin & Membrane Curvature
Q: What is cardiolipin?
A: A mitochondrial phospholipid essential for membrane bending & cristae formation.
Q: How does cardiolipin shape membranes?
A: Its dimeric, conical structure induces curvature, binds proteins, and stabilizes high-curvature regions.
Ubiquitin & Ubiquitin-Like (UBL) Signalling
Q: What is ubiquitin-type signalling?
A: A post-translational modification (PTM) where proteins are tagged with UBL peptides, affecting function, localization, or degradation.
UBL Enzymes
Q: What enzymes add UBL tags?
A: E1 (activation) → E2 (conjugation) → E3 (ligation) → E4 (extension/branching).
Q: What enzymes remove UBL tags?
A: Deubiquitinases (DUBs)—thiol proteases & metalloproteases.
UBL Attachment & Chain Formation
Q: How is a UBL attached to a protein?
A: UBL (-COOH) + lysine (-NH₂) → isopeptide bond via a condensation reaction.
Q: How do UBL chains form?
A: Branched or linear (M1) chains via isopeptide linkages between ubiquitin molecules.
Functions of UBL Tagging
Q: What are the effects of UBL conjugation?
Controls protein levels
Changes protein localization
Alters protein function
Regulates gene transcription
Deubiquitinases (DUBs) & Their Roles
Q: What are the key functions of DUBs?
A:
Recycle free ubiquitin
Rescue proteins from degradation
Restore protein function
Q: What cellular processes are regulated by DUBs?
Proteasome (K48 chains) → protein degradation
Autophagy (ATG genes) → cell survival
Signalling → phosphorylation & gene expression
Trafficking (K63 chains) → endosomal sorting
DNA damage response (DDR)
Cell cycle (cyclins) → proliferation control
Apoptosis → programmed cell death
Ubiquitination & Degradation
Q: How does ubiquitination lead to degradation?
A: K48-linked chains target proteins for proteasomal degradation via ubiquitin receptors.
Ubiquitin & Cell Signalling
Q: How does ubiquitination regulate receptor tyrosine kinases (RTKs)?
A: EGFR & HER2 (regulate cell growth) undergo Ub-mediated internalization → affects downstream signalling & recycling.
Q: How does ubiquitination regulate nuclear localization?
A: Ub tags control nuclear import/export of transcription factors.
Ubiquitin in DNA Damage Response (DDR)
Q: How does ubiquitin regulate DNA repair?
A: Ub modifications recruit repair proteins & regulate damage checkpoints.
Ubiquitin in Cell Cycle Control
Q: How does ubiquitin regulate the cell cycle?
A: Cyclins & checkpoint proteins are ubiquitinated for timely degradation.
P53 Ubiquitination & SUMOylation
Q: How is p53 regulated?
A: MDM2 (E3 ligase) ubiquitinates p53 for degradation, controlling cell proliferation.
Q: How are p53 phosphorylation & ubiquitination linked?
A: Phosphorylation can enhance or prevent p53 ubiquitination, affecting stability.
Ubiquitin Ligase Dysfunction in Disease
Q: How does defective ubiquitin ligase activity contribute to disease?
A: Cancer, cardiovascular disease (CVD), & neurodegeneration arise from faulty protein degradation.
Therapeutic Targeting of UBL Enzymes
Q: How can UBL enzymes be targeted for therapy?
A: Inhibitors/modulators of E3 ligases, DUBs, & proteasomal pathways are potential drug targets.
Secretory Protein Entry into ER
Q: How do secretory proteins enter the ER lumen?
A: Signal peptidase cleaves the signal sequence, allowing the polypeptide to pass through the translocon into the ER lumen.
Signal Peptidase (SPase)
Q: What is the role of signal peptidase?
A: Cleaves signal peptides of soluble & Type I proteins in the ER lumen.
Q: Why aren’t all signal sequences cleaved?
A: Some lack a specific cleavage site or have large/charged side chains.
Driving Force for ER Translocation
Q: What drives the unidirectional movement of proteins into the ER?
A: Sec63 complex + BiP (HSP chaperone)—BiP binds & stabilizes the growing polypeptide, preventing backsliding.
Q: How does Sec63 regulate BiP?
A: Promotes BiP-ATP hydrolysis, causing BiP to bind incoming polypeptides.
ER Membrane Protein Insertion
Q: What are the key features of integral membrane proteins?
A: They remain embedded in the membrane bilayer and follow the same translocation pathway as soluble proteins.
Type I Integral Membrane Proteins
Q: How do Type I membrane proteins insert?
Signal peptidase cleaves the N-terminal signal sequence.
Hydrophobic TM domain (~20 aa, α-helix) enters translocon.
Stop-Transfer Anchor (STA) sequence halts translocation.
TM domain embeds into the ER membrane; C-terminus remains cytosolic.
Type II & Type III Membrane Proteins
Q: How do Type II & III membrane proteins differ from Type I?
Type II: No cleavable signal, C-terminal in ER lumen, N-terminal cytosolic.
Type III: Same topology as Type I but no cleavable signal.
Key difference: Type II (+) charges on N-terminal side, Type III (+) charges on C-terminal side.
Type IV Membrane Proteins
Q: What defines Type IV membrane proteins?
A: Multiple TM domains inserted sequentially via translocon lateral gating.
Tail-Anchored Membrane Proteins
Q: How do tail-anchored proteins insert?
A: C-terminal hydrophobic tail directs them to the ER post-translationally.
Predicting Membrane Protein Topology
Q: How does a hydropathy profile predict topology?
A: Assigns + values to hydrophobic & – values to hydrophilic amino acids, identifying signal sequences & TM domains.
GPI-Anchored Proteins
Q: What is a GPI anchor, and how is it attached?
A: Glycosylphosphatidylinositol (GPI) covalently links proteins to the membrane via transamidase cleavage & transfer.
Post-Translational Modifications (PTMs) in the Secretory Pathway
Q: What PTMs occur during secretion?
N-linked glycosylation (ER)
Disulfide bond formation (ER)
Protein oligomerization (ER)
Golgi modifications
N-Linked Glycosylation
Q: Where does N-linked glycosylation occur?
A: In the ER lumen, on asparagine (N) residues.
Disulfide Bond Formation
Q: Where do disulfide bonds form?
A: In the oxidizing environment of the ER lumen, between cysteine residues.
ER Quality Control & Degradation
Q: What happens to misfolded proteins?
A: Tagged with ubiquitin & sent to the proteasome for degradation.
Golgi Targeting & Modification
Q: What signals retain proteins in the ER vs. send them to the Golgi?
KDEL sequence → ER retention
TMD targeting signals → Golgi sorting
Golgi Sorting & Trafficking
Q: What happens at the Golgi during secretion?
A: Sorting, glycosylation, & modification before vesicle transport.
Vesicle-Mediated Transport
Q: What is the role of clathrin in vesicle sorting?
A: Clathrin triskelion forms a coated vesicle for TGN-to-PM trafficking.
Q: What protein facilitates vesicle uncoating?
A: Dynamin—mediates vesicle scission.
Mannose-6-Phosphate (M6P) Sorting
Q: What is the role of M6P?
A: Targets lysosomal enzymes to the lysosome.
Endocytosis Pathways
Q: What are the three main endocytosis pathways?
Clathrin-mediated endocytosis (receptor-specific)
Caveolin-mediated endocytosis (lipid raft-dependent)
Macropinocytosis (non-specific fluid uptake)
Mitochondrial Targeting Signals (MTS)
Q: What are mitochondrial targeting signals (MTS)?
A: Amphipathic α-helices at the N-terminus of precursor proteins, directing them to mitochondria.
Q: What characteristics define MTS sequences?
15-50 amino acids long
Positively charged residues (Arg, Lys) on one side
Hydrophobic residues on the other side
Recognized by TOM (Translocase of the Outer Membrane)
Mitochondrial Import Pathway
Q: How do proteins enter mitochondria?
Synthesized in the cytosol as precursor proteins
Chaperones (Hsp70, Hsp90) prevent premature folding
TOM complex recognizes & transports across the outer membrane
TIM complex inserts proteins into the inner membrane or translocates them into the matrix
Outer Membrane Proteins
Q: How do proteins integrate into the outer membrane?
Beta-barrel proteins (e.g., porins) use the SAM complex
Single-pass α-helical proteins use the Mim1 complex
TOM complex assists initial insertion
Inner Membrane Proteins
Q: How are inner membrane proteins targeted?
A: Three pathways:
Stop-Transfer Pathway – Uses TIM23, embeds proteins with a stop-transfer sequence.
OXA Pathway – Precursor enters the matrix, then is inserted into the inner membrane via OXA1.
Carrier Pathway – Small metabolites transporters (e.g., ATP/ADP carriers) use TIM22 complex.
Mitochondrial Matrix Proteins
Q: How do proteins reach the mitochondrial matrix?
A:
MTS recognized by TOM complex.
Translocated through TIM23.
Mitochondrial Hsp70 pulls protein into matrix.
MTS is cleaved by Mitochondrial Processing Peptidase (MPP).
Protein folds with the help of Hsp60 chaperones.
Nuclear Protein Targeting
Q: What directs proteins to the nucleus?
A: Nuclear Localization Signals (NLS)
Short, basic amino acid sequences (Lys, Arg-rich)
Recognized by importins for nuclear import
Transported through the Nuclear Pore Complex (NPC)
Q: What is the mechanism of nuclear import?
Importin-α binds NLS of cargo protein.
Importin-β mediates translocation through the NPC.
Ran-GTP binding releases the protein inside the nucleus.
Importins return to the cytoplasm for reuse.
Q: What is nuclear export?
A: Uses Nuclear Export Signals (NES), recognized by exportins.
Peroxisome Targeting
Q: How are proteins targeted to peroxisomes?
A: Two main signals:
PTS1: C-terminal (Ser-Lys-Leu, SKL motif)
PTS2: N-terminal (9-amino acid sequence)
Q: How do proteins enter peroxisomes?
PTS1/PTS2 sequences recognized by receptors (Pex5/Pex7).
Pex receptors escort proteins to peroxisome membrane.
Proteins enter via the Pex complex using ATP.
Receptors recycled back to the cytosol.