Cell Biology: Rab Proteins in Vesicle Trafficking

Vesicle Trafficking

• Proteins are packed into vesicles for trafficking from the ER.
• Rab proteins, a type of small G-protein, serve as identity labels for organelles.
• Rab is active when bound to GTP and inactive when bound to GDP.

Rab Activation and Inactivation

• Rab-GDP is recruited to intracellular membrane.
• Activation occurs when the host membrane has specific proteins to form Rab-GTP, allowing proof-reading.
• Recognition of Rab-GTP by another organelle is stabilized if it can hydrolyze Rab-GTP to form Rab-GDP.
• Rab-GDP is then released from the membrane to be re-used.

Diseases Caused by Defective Rabs

• Charcot Marie tooth disease 2B is caused by a Rab7 mutation.
• Legionnaire's disease is caused by a Rab1 mutation.

Protein Trafficking

• Proteins folded and N-glycosylated in the ER are collected into COPII vesicles.
• COPII vesicles move to the cis Golgi, where sugar structures of proteins are trimmed/modified.
• For proteins directed to lysosomes, mannose-6-phosphate (M6P) is attached.

COPII Vesicle Formation

• COPII proteins bind to a specific cytosolic sequence of proteins embedded in the ER membrane.
• They collect proteins into clusters with attached luminal proteins, which are then pinched off as small COPII vesicles.
• COPII vesicles move along microtubules to the cis Golgi, shedding their coat on the way.

Organelle Fusion

• SNARE (SNAP receptor) proteins mediate organelle fusion, drawing membranes close together.
• COPII vesicles must fuse with cis Golgi only, making recognition important.

COPI Vesicle Formation

• COPI vesicles move proteins from the Golgi to the ER.
• Luminal cargoes are recognized by a KDEL sequence, which binds to the KDEL receptor on the Golgi membrane.
• The COPI coat assembles, and subsequent events occur in reverse as the COPII vesicle mechanism.

Protein Trafficking from the Golgi

• Soluble proteins destined for lysosomes are recognized by the M6P modification acquired in the Golgi.
• They bind to the M6P receptor, which recognizes M6P at the luminal surface.
• They have short sequences that allow assembly of a clathrin coat at the cytosolic surface.
• Coated vesicles punch off from the Golgi, are transported, uncoated, and recognized by late endosomes, which they fuse with.

Lysosomal Functions

• Low pH causes the cargo to dissociate from the M6P receptor, which is transported back to the trans-Golgi.
• The cargo proceeds to a lysosome, where enzymes degrade imported materials and recycle macromolecules.
• Genetic differences in these enzymes underlie a variety of lysosomal storage diseases (LSDs).

Enzyme Replacement Therapy

• Imperfect targeting of M6P receptors allows some to escape to the plasma membrane, where they can be recovered to lysosomes by endocytosis.
• Administration of M6P-modified forms of missing enzymes in Gaucher disease results in the enzyme being endocytosed by the M6P receptor and delivered to lysosomes.

LDL Receptor Trafficking

• All cells express low-density lipoprotein (LDL) receptors, which bind to the ApoB-100 component of LDL.
• The cytosolic domain of the LDL receptor has sequences that interact with AP2 and thereby clathrin, which assembles LDL receptors and cargo into clathrin-coated pits for trafficking to endosomes.
• In the acidic lumen of endosomes, LDL dissociates from its receptor.

Ion Channels

• Ion channels open and close quickly, in less than 1 ms, and can allow several million ions to pass through.
• They mediate the fastest communication in the cell.

Regulation of Intracellular Activities

• Membrane potential changes are transduced as a messenger that enters the cell, typically calcium, through the opening of voltage-gated calcium channels (VGCaCs).
• VGNa/KCs are analogous to cables, while VGCaCs are analogous to the appliance, and VGICs are important therapeutic targets.

Nicotinic Acetylcholine Receptors

• Nicotinic acetylcholine receptors, found in muscle, respond to acetylcholine (ACh) released from pre-synaptic fibers.
• Drugs interacting with these receptors can relax muscle (during surgery) and treat myasthenia gravis (an autoimmune condition).

GABA/Glycine Receptors

• GABA/glycine receptors, which are chloride permeable, mediate most synaptic transmission in the CNS.
• Drugs targeted to these receptors are used to relieve anxiety and as general anesthetics.

Voltage-Gated Cation Channels

• Voltage-gated cation channels consist of 24 membrane spanning regions (4 × 6) arranged around a central pore.
• For Na+ and Ca2+ channels, the entire channel is comprised of a single protein chain.
• For K+ channels, four protein chains, each with six membrane-spanning domains, are required.

Ion Selectivity

• Ion selectivity arises from a narrow selectivity filter, where cations pass through single file, and surrounding amino acid residues interact.
• The selectivity filter of Ca2+ channels has two Ca2+ binding sites, which can bind Ca2+ selectively and tightly, allowing selectivity.

Channel Gating

• In closed channels, the residues at the tip of TMD6 come together to form a tepee shape, occluding the channel.
• The voltage sensor consists of four positively charged residues on TMD4, which move outwards in the plane of the membrane when the PM depolarizes.
• This movement pulls a short cytosolic helix linking TMD4-5, which links the voltage-sensor to TMDs forming the pore, leading to twisting of pore-lining helices and opening of the channel.