UnitA 4

• The Endoplasmic Reticulum (ER) is a network of membrane-enclosed tubules and sacs (cisternae) that extends from the nuclear membrane throughout the cytoplasm and is the largest membrane-bound organelle present in eukaryotic cells.
• The ER is the primary site for protein synthesis, folding and transport (rough ER) as well as lipid synthesis (smooth ER) & calcium storage.
• The ER serves as the entry point for proteins destined for the Golgi Apparatus (GA), endosomes, lysosomes & cell surface.
• The ER and the Plasma Membrane (PM) communicate via membrane contact sites (MCS).
• Proteins are transferred from the cytosol to the ER include water-soluble proteins destined for secretion from the cell or lumen of an endomembrane organelle and transmembrane proteins that remain in the membrane of an endomembrane organelle or the PM.
• The ER is the sorting site for many cellular proteins.
• A common pool of ribosomes is responsible for the synthesis of non-ER and ER-bound proteins.
• There is a free ribosome cycle where ribosomes synthesizing proteins lacking ER signal are used.
• There is also a membrane-bound ribosome cycle where synthesis of proteins containing a signal sequence for the ER occurs.
• COPII vesicles bud from the ER and carry their cargo proteins forward to the cis-Golgi.
• Each of these complexes is composed of two large adaptins (one each of γ/α/δ/ε and β1-4, respectively, 90-130 kDa), one medium adaptin (μ1-4, ∼50 kDa), and one small adaptin (ς1-4, ∼20 kDa).
• Adaptins have 21-83% identity similarity at the amino acid level between complex subunits.
• The purpose of adaptins is to tether clathrin to the cargo receptor on the surface of vesicles.
• Clathrin-adaptin molecules are then released and the naked vesicle fuses to the target membrane.
• A vesicle starts as a clathrin-coated "pit" with adaptins binding the cargo receptor with its cargo molecule.
• Dynamin (GTP-binding protein) causes ring constriction and the clathrin-coated vesicle buds off.
• Four types of adaptin complexes were isolated in 1975: AP-1, AP-2, AP-3 and AP-4.
• SNARE proteins on the vesicle (v-SNARES) will also bind SNARES present on the target membrane for additional anchoring support.
• Rab (GTPase) proteins marked on each vesicle are recognized by tethering proteins on the cytosolic surface of the target membrane.
• Complexes composed of subunits around 100 kDa in size.
• Several adaptins then bind clathrin at the cytosolic surface of the plasma membrane resulting in invagination of the clathrin-coated pit.
• Once a transport vesicle buds, it must make its way to its destination.
• In flies, dynamin mutations fail in the budding of clathrin-coated vesicles, resulting in paralysis.
• Such an arrangement is referred to as a trans-SNARE complex since the SNAREs are on two distinct membranes.
• Three helices are contributed by the t-SNARE proteins and one helix is contributed by the v-SNARE.
• When a vesicle is docked, the SNAREs associate as a bundle of 𝛂-helices called the 4-helix bundle.
• Adaptor complexes help link Clathrin to the membrane bilayer.
• Vesicular transport is highly organized: the membrane of each vesicle maintains its orientation.
• Membrane cargo in the ER can be recognized by cytosolic proteins that will aid in vesicle formation.
• The exocytic pathway involves proteins being synthesized on the ER membrane, entering the ER, going to the Golgi apparatus, reaching the cell surface, and ending in endosomes and lysosomes.
• Proteins released from the endoplasmic reticulum (ER) enter the Golgi apparatus where they are sorted and modified.
• Clathrin is a molecular scaffold protein that forms a lattice-like coat on and around membranes, composed of trimer of three (3) heavy and light polypeptide chains and 4 adaptor complexes.
• Vesicle budding is driven by protein coating of the cytoplasmic surfaces of transport vesicles, with three (3) types of coated vesicles: Clathrin-coated, COP I-coated, and COP II-coated.
• Quality control in the ER involves checking if the protein is folded and if the protein complex is assembled, and if not, it is actively retained by ER-localized chaperones.
• Exocytosis is the process where proteins/lipids fuse to the plasma membrane and are released to the extracellular space.
• Active cargo selection in the ER involves specific cargo being collected in regions of the ER that will pinch off to form a transport vesicle.
• Mannose - 6 - phosphate directs proteins to the lysosome.
• The endocytic pathway involves the ingestion of extracellular material at the plasma membrane, going to endosomes, reaching lysosomes, and ending in degradation.
• Endocytosis is the process where extracellular material is engulfed into vesicles that bud inward.
• Soluble cargo in the ER are recognized by membrane proteins that span the ER bilayer.