Unit 2: Cellular Transport and Trafficking; Cell Cycle and its Regulation - Subjective Questions

Passive Transport:

• Movement of molecules across the membrane without expenditure of metabolic energy.
• Occurs down the concentration gradient (high to low concentration).
• Includes simple diffusion, facilitated diffusion, and osmosis.
• Example: Diffusion of and across membranes.

Active Transport:

• Movement of molecules against the concentration gradient (low to high concentration).
• Requires energy, usually in the form of ATP hydrolysis (primary active transport) or an ion gradient (secondary active transport).
• Mediated by specific carrier proteins/pumps.
• Example: -ATPase pump.

Key Differences:

The -ATPase pump is a primary active transporter present in the plasma membrane of animal cells.

Structure:

• It is a tetramer composed of two -subunits (catalytic) and two -subunits (regulatory/glycoprotein).
• The -subunit contains binding sites for , , and ATP.

Mechanism (Cycle):

1. Three ions from the cytoplasm bind to the pump.
2. ATP is hydrolyzed, phosphorylating the pump (aspartate residue).
3. Conformational change (E1 → E2) releases the 3 outside the cell.
4. Two ions from outside bind to the pump.
5. Dephosphorylation causes reversion to the original conformation (E2 → E1).
6. The 2 ions are released into the cytoplasm.

Overall: For every ATP hydrolyzed, 3 out and 2 in.

Importance:

• Maintains the resting membrane potential (electrogenic pump).
• Regulates cell volume and osmotic balance.
• Drives secondary active transport of glucose and amino acids.
• Essential for nerve impulse conduction and muscle contraction.

These are types of membrane transport by carrier proteins based on the number and direction of solutes moved.

Uniport:

• Transports a single type of solute in one direction.
• Example: GLUT transporters for glucose.

Symport (Cotransport):

• Transports two different solutes in the same direction simultaneously.
• Example: -glucose symporter (SGLT) in intestinal epithelial cells.

Antiport (Exchanger):

• Transports two solutes in opposite directions.
• Example: exchanger and antiporter.

Comparison Table:

Both symport and antiport are examples of coupled transport (secondary active transport), where the energy from one gradient drives the movement of another.

The Nuclear Pore Complex (NPC) is a large protein assembly that perforates the nuclear envelope and mediates transport between the nucleus and cytoplasm.

Structure:

• One of the largest protein complexes in the cell (~125 MDa in vertebrates).
• Composed of multiple copies of ~30 different proteins called nucleoporins (Nups).
• Displays octagonal symmetry.
• Consists of: cytoplasmic filaments, cytoplasmic ring, central channel, nuclear ring, and a nuclear basket.
• The central channel contains FG-nucleoporins (rich in phenylalanine-glycine repeats) that form a selective barrier.

Functions:

• Passive diffusion of small molecules and ions (< 40 kDa).
• Active, selective transport of large molecules (proteins, RNA) requiring signals:
  • Nuclear Localization Signal (NLS) for import.
  • Nuclear Export Signal (NES) for export.
• Transport is mediated by karyopherins (importins/exportins) and regulated by the Ran-GTPase gradient.
• Acts as a selective gatekeeper controlling gene expression and cellular signaling.

The Ran-GTPase is a small G-protein that provides directionality and energy to nucleocytoplasmic transport.

The Ran Gradient:

• Ran-GTP is concentrated in the nucleus (due to the chromatin-bound GEF called RCC1).
• Ran-GDP predominates in the cytoplasm (due to the cytoplasmic GAP called RanGAP).

Nuclear Import:

1. A cargo protein with an NLS binds importin in the cytoplasm.
2. The complex translocates through the NPC into the nucleus.
3. Ran-GTP binds importin, causing cargo release in the nucleus.
4. Importin–Ran-GTP recycles back to the cytoplasm.

Nuclear Export:

1. Exportin binds cargo (containing NES) and Ran-GTP in the nucleus.
2. The complex moves to the cytoplasm.
3. RanGAP stimulates GTP hydrolysis → Ran-GDP, releasing the cargo.
4. Exportin recycles into the nucleus.

Significance:

• The GTP/GDP conformational switch ensures unidirectional transport.
• The steep Ran-GTP gradient across the envelope powers transport.
• Energy is derived from GTP hydrolysis.

Most mitochondrial proteins are encoded by nuclear genes, synthesized on cytosolic ribosomes, and imported post-translationally.

Steps of Import into the Matrix:

1. Precursor protein synthesis: Proteins are made with an N-terminal presequence (matrix-targeting signal) — an amphipathic helix rich in positively charged residues.
2. Chaperone binding: Cytosolic Hsp70 keeps the precursor unfolded.
3. Recognition: The precursor binds receptors of the TOM complex (Translocase of the Outer Membrane).
4. Outer membrane crossing: Passage through the TOM channel.
5. Inner membrane crossing: Transfer to the TIM23 complex (Translocase of the Inner Membrane).
   • Driven by the membrane potential () across the inner membrane.
6. Pulling into the matrix: Matrix Hsp70 (mtHsp70) acts as an import motor, using ATP hydrolysis to pull the protein in.
7. Processing: The matrix processing peptidase (MPP) cleaves off the presequence.
8. Folding: Chaperonins (Hsp60/Hsp10) fold the mature protein.

Energy sources: ATP hydrolysis (cytosolic and matrix Hsp70) and the electrochemical gradient ().

Both organelles import most of their proteins from the cytosol post-translationally, but there are important differences.

Similarities:

• Proteins synthesized on cytosolic ribosomes and imported post-translationally.
• Both use N-terminal targeting signals (cleavable).
• Require cytosolic chaperones (Hsp70) to keep precursors unfolded.
• Use translocase complexes in outer and inner membranes.

Differences:

Chloroplast-specific: Proteins destined for the thylakoid lumen require a second targeting sequence revealed after the transit peptide is removed in the stroma.

After a protein reaches the stroma and its transit peptide is cleaved, a second signal directs it into the thylakoid lumen via one of four pathways:

1. Sec (Secretory) Pathway:

• Transports unfolded proteins.
• Uses SecA (ATPase) and the SecYEG translocon.
• Energy from ATP hydrolysis.

2. Tat (Twin-Arginine Translocation) Pathway:

• Transports fully folded proteins.
• Recognizes a twin-arginine (RR) motif in the signal.
• Uses the proton gradient () as the driving force.

3. SRP (Signal Recognition Particle) Pathway:

• For integral thylakoid membrane proteins (e.g., light-harvesting proteins).
• Uses a chloroplast SRP (cpSRP) and GTP.

4. Spontaneous Pathway:

• Some proteins insert directly into the membrane without translocases or energy.

Summary: The choice of pathway depends on the folding state and the nature of the signal sequence, reflecting the endosymbiotic bacterial origin of chloroplasts.

The secretory pathway transports proteins from their site of synthesis to their final destinations via membrane-bound vesicles.

Steps:

1. Synthesis at the ER: Secretory and membrane proteins are synthesized on ribosomes bound to the rough ER; the signal sequence directs them into the ER lumen (co-translational translocation via the SRP/translocon).
2. Folding & modification: Proteins fold with the help of chaperones; N-linked glycosylation begins here.
3. ER to Golgi transport: Correctly folded proteins are packaged into COPII-coated vesicles and transported to the ERGIC, then to the cis-Golgi.
4. Golgi processing: Proteins move cis → medial → trans through the Golgi, undergoing further glycosylation, sulfation, and phosphorylation.
5. Sorting at the trans-Golgi network (TGN): Proteins are sorted into vesicles for various destinations.
6. Exocytosis: Vesicles fuse with the plasma membrane to release contents.
   • Constitutive secretion: Continuous, unregulated (e.g., collagen).
   • Regulated secretion: Vesicles stored until a signal (e.g., ) triggers release (e.g., hormones, neurotransmitters).

Retrograde transport: COPI-coated vesicles return proteins from Golgi to ER, maintaining compartment identity.

Coated vesicles are the primary carriers in vesicular trafficking, each with distinct coats and functions.

COPII-coated vesicles:

• Mediate anterograde transport from the ER → Golgi.
• Coat proteins: Sar1-GTP, Sec23/24, Sec13/31.
• Package correctly folded cargo, leaving out ER-resident proteins.

COPI-coated vesicles:

• Mediate retrograde transport from Golgi → ER and intra-Golgi transport.
• Coat proteins: ARF1-GTP and coatomer complex.
• Retrieve escaped ER proteins bearing the KDEL/KKXX retrieval signals.

Clathrin-coated vesicles:

• Mediate transport from the plasma membrane (endocytosis) and from the trans-Golgi network → endosomes/lysosomes.
• Coat proteins: Clathrin triskelions + adaptor proteins (AP complexes).
• Important for receptor-mediated endocytosis.

Common features:

• Coat assembly is regulated by small GTPases (Sar1, ARF).
• Coats provide the mechanical force to deform membranes and select cargo, then are shed before fusion.

SNAREs (Soluble NSF Attachment protein REceptors) are membrane proteins that ensure specificity of vesicle docking and fusion.

Types:

• v-SNAREs (vesicle SNAREs): Present on the transport vesicle (e.g., synaptobrevin/VAMP).
• t-SNAREs (target SNAREs): Present on the target membrane (e.g., syntaxin and SNAP-25).

Mechanism:

1. Tethering: Rab-GTPases and tethering proteins bring the vesicle close to the target membrane.
2. Docking: The complementary v-SNARE and t-SNARE recognize each other.
3. Trans-SNARE complex formation: They coil around each other forming a tight four-helix bundle, pulling the two membranes together.
4. Fusion: This close apposition forces the lipid bilayers to fuse, releasing the cargo.
5. Recycling: After fusion, the NSF (an ATPase) and its adaptor α-SNAP disassemble the SNARE complex using ATP hydrolysis, recycling SNAREs.

Significance:

• Provides specificity — each vesicle fuses only with its correct target.
• Central to processes like neurotransmitter release; targeted by toxins (e.g., botulinum and tetanus toxins cleave SNAREs).

Lysosomal enzymes (acid hydrolases) are sorted by a specific mannose-6-phosphate (M6P) tagging system.

Steps:

1. Synthesis: Lysosomal hydrolases are synthesized in the rough ER and receive N-linked oligosaccharides.
2. Tag addition in cis-Golgi: The enzyme GlcNAc phosphotransferase recognizes a signal patch on lysosomal enzymes and adds GlcNAc-phosphate to mannose residues; a second enzyme removes GlcNAc, exposing mannose-6-phosphate (M6P).
3. Recognition in TGN: M6P receptors in the trans-Golgi network bind the M6P-tagged enzymes.
4. Vesicle packaging: Receptor–enzyme complexes are packaged into clathrin-coated vesicles.
5. Delivery to late endosome: Vesicles fuse with late endosomes; the low pH (~6) causes the enzyme to dissociate from the receptor.
6. Receptor recycling: M6P receptors return to the TGN for reuse.
7. Maturation: The M6P group is removed, and the endosome matures into a lysosome.

Clinical significance: Defects in this pathway cause I-cell disease (Mucolipidosis II), where enzymes are secreted instead of being delivered to lysosomes.

The cell cycle is the ordered sequence of events by which a cell grows, duplicates its DNA, and divides into two daughter cells.

Phases:

1. Interphase (period between divisions; ~90% of cycle):

• phase (Gap 1): Cell grows, synthesizes proteins and organelles; monitors readiness for DNA synthesis.
• S phase (Synthesis): DNA replication occurs; each chromosome is duplicated into two sister chromatids.
• phase (Gap 2): Cell continues to grow, synthesizes proteins for mitosis, and checks DNA for errors.

2. M phase (Mitotic phase):

• Mitosis: Division of the nucleus (prophase, prometaphase, metaphase, anaphase, telophase).
• Cytokinesis: Division of the cytoplasm into two daughter cells.

phase:

• A resting/quiescent state where cells exit the cycle (e.g., neurons, mature muscle cells).

Regulation: The cycle is controlled at checkpoints (, , and spindle assembly checkpoint) by cyclins and cyclin-dependent kinases (CDKs).

The cell cycle is driven by cyclin–CDK complexes that act as molecular switches.

Cyclin-Dependent Kinases (CDKs):

• Serine/threonine protein kinases whose levels remain constant through the cycle.
• Inactive on their own; require binding to a cyclin to become active.

Cyclins:

• Regulatory proteins whose concentration oscillates (synthesized and degraded) during the cycle.
• Different cyclins are expressed at specific phases.

Major complexes:

• Cyclin D–CDK4/6: Progression through early .
• Cyclin E–CDK2: transition.
• Cyclin A–CDK2: S phase progression.
• Cyclin B–CDK1 (MPF): transition (mitosis).

Regulation of CDK activity:

1. Cyclin binding (activation).
2. Phosphorylation/dephosphorylation by CAK and Wee1/Cdc25.
3. CDK inhibitors (CKIs) such as p21 and p27.
4. Cyclin degradation via ubiquitin-proteasome pathway (APC/C).

Significance: Cyclin–CDK complexes phosphorylate target proteins (e.g., Rb protein) to trigger transitions and ensure events occur in the correct order.

Checkpoints are surveillance mechanisms that ensure each phase is completed accurately before the next begins, preventing propagation of errors.

1. Checkpoint (Restriction Point):

• The most important checkpoint in mammalian cells.
• Checks for cell size, nutrients, growth factors, and DNA damage.
• If DNA is damaged, p53 halts the cycle and activates p21 (a CDK inhibitor).
• Determines whether the cell commits to division or enters .

2. Checkpoint:

• Ensures DNA replication is complete and DNA is undamaged before mitosis.
• Controlled by Cyclin B–CDK1 (MPF) activation via Cdc25 phosphatase.

3. Spindle Assembly Checkpoint (Metaphase-to-Anaphase / M checkpoint):

• Ensures all chromosomes are correctly attached to spindle microtubules at the metaphase plate.
• Prevents anaphase until proper attachment; blocks APC/C activation.
• Prevents aneuploidy.

Significance:

• Maintain genomic integrity.
• Prevent transmission of damaged DNA.
• Failure of checkpoints (e.g., p53 mutation) can lead to cancer.

Mitosis is the division of the nucleus resulting in two genetically identical daughter nuclei. It occurs in the following stages:

1. Prophase:

• Chromatin condenses into visible chromosomes (each with two sister chromatids).
• The mitotic spindle begins forming from the centrosomes.
• The nucleolus disappears.

2. Prometaphase:

• The nuclear envelope breaks down.
• Spindle microtubules attach to chromosomes at the kinetochores.

3. Metaphase:

• Chromosomes align at the metaphase plate (equatorial plane).
• Spindle assembly checkpoint ensures all kinetochores are attached.

4. Anaphase:

• Sister chromatids separate (cohesin cleaved by separase) and move to opposite poles.
• Anaphase A: chromatids move to poles; Anaphase B: poles move apart.

5. Telophase:

• Chromosomes reach the poles and begin to decondense.
• Nuclear envelopes reform around each set.
• Nucleolus reappears; spindle disassembles.

Followed by Cytokinesis: Division of the cytoplasm producing two diploid daughter cells, each genetically identical to the parent.

Both are forms of nuclear division, but they serve different biological purposes.

Key points:

• Mitosis maintains chromosome number and produces cells for growth and repair.
• Meiosis reduces chromosome number by half and introduces genetic variation through crossing over and independent assortment.
• Meiosis is essential for sexual reproduction and maintaining constant chromosome number across generations.

Meiosis generates genetic diversity through two major mechanisms.

1. Crossing Over (Recombination):

• Occurs during Prophase I (pachytene stage).
• Homologous chromosomes pair up (synapsis) forming a bivalent/tetrad.
• Non-sister chromatids exchange segments at points called chiasmata.
• This produces recombinant chromatids with new combinations of alleles.

2. Independent Assortment:

• Occurs during Metaphase I.
• Homologous pairs align randomly at the metaphase plate.
• The orientation of each pair is independent of others.
• Number of possible combinations = , where is the number of chromosome pairs.
• In humans (), this gives million combinations.

Significance:

• Both mechanisms shuffle alleles, creating genetically unique gametes.
• Combined with random fertilization, this produces enormous variation.
• Genetic variation is the raw material for evolution and natural selection.
• Enables populations to adapt to changing environments.

Cytokinesis is the division of the cytoplasm following nuclear division, but the mechanism differs between animal and plant cells due to the presence of a cell wall in plants.

Cytokinesis in Animal Cells:

• Occurs by cleavage furrow formation.
• A contractile ring of actin and myosin filaments forms beneath the plasma membrane at the equator.
• The ring contracts like a purse-string, pinching the cell inward.
• Cell divides from the outside inward (centripetal).

Cytokinesis in Plant Cells:

• Occurs by cell plate formation.
• Golgi-derived vesicles carrying cell wall material align at the center forming the phragmoplast.
• Vesicles fuse to form a cell plate that grows outward.
• Cell divides from the inside outward (centrifugal).
• Eventually forms a new cell wall and plasma membrane.

Comparison Table:

Receptor-mediated endocytosis (RME) is a selective process by which cells internalize specific macromolecules bound to cell-surface receptors.

Mechanism:

1. Ligand binding: Specific ligands (e.g., LDL, hormones) bind to their cell-surface receptors.
2. Clustering: Receptor–ligand complexes cluster in clathrin-coated pits on the plasma membrane.
3. Invagination: The pit invaginates, aided by adaptor proteins (AP2) and clathrin.
4. Vesicle formation: The GTPase dynamin pinches off the vesicle, forming a clathrin-coated vesicle.
5. Uncoating: The clathrin coat is shed, forming an early endosome.
6. Sorting: In the acidic endosome, ligand dissociates from the receptor:
   • Receptors are usually recycled back to the membrane.
   • Ligands are directed to lysosomes for degradation.

Example — LDL uptake (Cholesterol):

• LDL particles bind LDL receptors and are internalized.
• In the endosome, LDL dissociates and is delivered to lysosomes, releasing cholesterol.
• Receptors are recycled.
• Defective LDL receptors cause familial hypercholesterolemia.

Significance: Enables efficient, specific uptake of nutrients, hormones, and signaling molecules.