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

The eukaryotic cell is a set of membrane-bounded compartments, and almost every process in this unit follows from the fact that the lipid bilayer is selectively permeable. Transport, sorting and division all exist to move molecules across or between these compartments while preserving their distinct chemistries.

• Selective permeability: Small nonpolar molecules (O₂, CO₂) cross freely; ions and polar solutes require protein transporters.
• Electrochemical gradient: The driving force on a solute combines its concentration gradient and, for ions, the membrane potential (Vₘ ≈ −60 to −70 mV inside).
• Compartment identity: Each organelle keeps a characteristic lumen (ER Ca²⁺ store, acidic lysosome ~pH 4.7, oxidising ER vs reducing cytosol).
• Signal-based sorting: Proteins carry address tags (signal sequences, NLS, KDEL, mannose-6-phosphate) read by dedicated receptors.
• Energy coupling: Directional movement uses ATP hydrolysis, GTP hydrolysis, or a pre-existing ion gradient.

II. Molecular Mechanisms of Membrane Transport

Movement of solutes across the plasma membrane and organelle membranes.

A. Passive versus active transport

Transport is classified by whether it consumes energy and by the direction relative to the gradient.

1. Passive (facilitated) transport: Down the electrochemical gradient, no ATP.
   • Channels: Aqueous pores; e.g. the K⁺ channel selectivity filter (Gly-Tyr-Gly backbone carbonyls) conducts ~10⁸ ions/s.
   • Carriers (uniporters): Bind and undergo conformational change; e.g. GLUT1 moves glucose down its gradient.
2. Active transport: Against the gradient, requires energy.
   • Primary: ATP driven; the Na⁺/K⁺-ATPase exports 3 Na⁺ and imports 2 K⁺ per ATP, setting Vₘ.
   • Secondary: Uses an ion gradient; the Na⁺-glucose symporter (SGLT1) drags glucose uphill using the inward Na⁺ gradient.

B. Coupled transporters and pumps

Transporters are grouped by how many species move and in which direction.

• Uniport: One solute (GLUT family).
• Symport: Two solutes, same direction (Na⁺/glucose).
• Antiport: Two solutes, opposite directions (Na⁺/Ca²⁺ exchanger, 3 Na⁺ in per Ca²⁺ out).
• P-type pumps: Phosphorylate an aspartate residue during the cycle (Na⁺/K⁺-, Ca²⁺-ATPase).

The equilibrium potential of an ion is given by the Nernst equation:

E_ion = (RT / zF) · ln([ion]_out / [ion]_in)

• R = gas constant, T = temperature (K), z = ionic charge, F = Faraday constant.
• At 37 °C this reduces to E = (61.5/z) · log₁₀([out]/[in]) mV.

C. Significance

• Osmotic balance: The Na⁺/K⁺-ATPase prevents osmotic swelling and lysis.
• Electrical signalling: Ion channels underlie the action potential.

III. Nuclear Transport

Bidirectional exchange of macromolecules between nucleus and cytoplasm.

A. The nuclear pore complex (NPC)

The NPC is the sole gateway through the double nuclear envelope.

• Structure: ~30 nucleoporins arranged with eightfold symmetry; central channel lined with FG-repeat (Phe-Gly) domains forming a hydrophobic mesh.
• Size cut-off: Molecules < ~40 kDa diffuse freely; larger cargo needs active, signal-mediated transport.

B. Signals and carriers

Directional transport depends on address tags and the Ran-GTP gradient.

• NLS (nuclear localisation signal): Basic residues (e.g. SV40 large-T PKKKRKV) recognised by importin-α/β for import.
• NES (nuclear export signal): Leucine-rich sequence bound by exportin (CRM1) for export.
• Ran gradient: Ran-GTP is high in the nucleus (via chromatin-bound GEF, RanGEF/RCC1) and low in cytoplasm (via RanGAP).
  • Import: Ran-GTP binds importin in the nucleus, releasing cargo.
  • Export: Ran-GTP promotes exportin–cargo binding in the nucleus; hydrolysis in cytoplasm releases cargo.

IV. Transport Across Mitochondria and Chloroplasts

Post-translational import of nuclear-encoded proteins into organelles that arose by endosymbiosis.

A. Mitochondrial protein import

Proteins made in the cytosol are threaded through translocase complexes in an unfolded state.

• Presequence: N-terminal amphipathic matrix-targeting sequence, positively charged.
• TOM complex: Translocase of the Outer Membrane; general entry gate (TOM40 pore).
• TIM23 complex: Translocase of the Inner Membrane; imports matrix proteins.
• Driving forces: The inner-membrane potential (Δψ) pulls the positive presequence; matrix Hsp70 acts as an ATP-driven ratchet.
• Processing: MPP (matrix processing peptidase) cleaves the presequence.

B. Chloroplast protein import

A parallel system operates across the two envelope membranes.

• Transit peptide: N-terminal signal directing proteins to the stroma.
• TOC/TIC complexes: Translocons of the Outer/Inner Chloroplast envelope.
• Energy source: GTP and stromal ATP (not a membrane potential, unlike mitochondria).
• Extra step: Thylakoid-destined proteins carry a second signal for onward routing (Sec, Tat, SRP pathways).

V. Intracellular Vesicular Trafficking from ER through Golgi to Lysosomes/Cell Exterior

The secretory pathway: budding, targeting and fusion of transport vesicles between compartments.

A. Entry into the endoplasmic reticulum

Secretory and membrane proteins are inserted co-translationally.

• Signal sequence: N-terminal hydrophobic stretch recognised by SRP (signal recognition particle).
• Translocon: Sec61 channel; ribosome docks via the SRP receptor.
• ER processing: Signal-peptidase cleavage, N-linked glycosylation (Glc₃Man₉GlcNAc₂ core added), disulphide bond formation, chaperone-assisted folding (BiP, calnexin).

B. Vesicle coats and targeting machinery

Each transport step uses a specific coat and fusion apparatus.

• COPII: ER → Golgi anterograde budding (Sar1-GTP recruits Sec23/24, Sec13/31).
• COPI: Golgi → ER retrograde retrieval (recovers KDEL-tagged ER-resident proteins via the KDEL receptor).
• Clathrin: Golgi → endosome and plasma-membrane endocytosis; adaptor proteins (AP complexes) link cargo to the coat.
• Targeting: Rab-GTPases and tethers give specificity; v-SNARE (vesicle) pairs with t-SNARE (target) to force fusion; NSF/SNAP recycle SNAREs.

C. Transit through the Golgi apparatus

The Golgi modifies and sorts cargo across ordered cisternae.

• Polarity: cis face (receiving) → medial → trans face (shipping); trans-Golgi network (TGN) is the sorting hub.
• Modifications: Trimming and remodelling of N-linked sugars, O-linked glycosylation, sulphation.
• Lysosomal tag: Enzymes destined for lysosomes gain mannose-6-phosphate (M6P) in the cis-Golgi, recognised by the M6P receptor in the TGN.

D. Delivery to lysosomes

Acid hydrolases are diverted from the default secretory route.

• Routing: M6P-receptor vesicles bud (clathrin-coated) from the TGN and fuse with late endosomes.
• Release: The low endosomal pH (~6) dissociates enzyme from receptor; the receptor recycles.
• Maturation: Late endosome matures into the lysosome (~pH 4.7) holding >40 acid hydrolases.

E. Delivery to the cell exterior

Secretion completes the pathway at the plasma membrane.

1. Constitutive secretion: Continuous, unregulated flow (e.g. serum albumin, ECM proteins) — the default route.
2. Regulated secretion: Cargo concentrated into secretory granules, released only on a signal (e.g. insulin on a Ca²⁺ rise), via SNARE-mediated exocytosis.

VI. Cell Cycle and its Regulation: Mitosis, Meiosis and Cytokinesis

Ordered duplication and partition of the genome, driven by cyclin-dependent kinases.

A. Cell-cycle phases and control

The cycle alternates DNA synthesis with division, gated at checkpoints.

• Phases: G₁ → S (DNA replication) → G₂ → M (mitosis); G₀ is a resting exit.
• Cyclin–CDK engine: CDK activity requires a cyclin partner whose level oscillates.
  • G₁/S-CDK (cyclin E–CDK2): Commits to replication past the restriction point.
  • M-CDK (cyclin B–CDK1, "MPF"): Triggers mitotic entry.
• Checkpoints: G₁ (DNA damage, p53 → p21 inhibits CDK), G₂/M (replication complete), spindle-assembly checkpoint (all kinetochores attached before anaphase).
• Irreversibility: The APC/C ubiquitin ligase tags securin and cyclin B for destruction, driving anaphase onset.

B. Mitosis

Equational division producing two genetically identical diploid daughter nuclei.

• Prophase: Chromosomes condense; centrosomes separate and nucleate the spindle.
• Prometaphase: Nuclear envelope breaks down; kinetochores capture microtubules.
• Metaphase: Chromosomes align at the metaphase plate.
• Anaphase: Separase cleaves cohesin; sister chromatids move to opposite poles (anaphase A) as poles separate (anaphase B).
• Telophase: Chromosomes decondense; nuclear envelope reforms.

C. Meiosis

Reductional division halving the chromosome number to make gametes.

1. Meiosis I (reductional): Homologues pair (synapsis, synaptonemal complex), crossing over at chiasmata recombines alleles; homologues (not sisters) separate — this is what halves ploidy.
2. Meiosis II (equational): Sister chromatids separate, resembling mitosis, with no preceding S phase.

• Outcome: One diploid cell → four haploid cells; independent assortment plus recombination generate genetic diversity.

D. Cytokinesis

Physical division of the cytoplasm following nuclear division.

1. Animal cells: An actin-myosin contractile ring pinches an inward cleavage furrow.
2. Plant cells: Golgi-derived vesicles fuse at the midline to build a cell plate that becomes the new cell wall.

• Positioning: The mitotic spindle specifies the division plane via RhoA activation at the equator.