Question

Detail methods of vesicular transport. Include in your discussion: clathrin, COPI, COPII, triskelion, cargo receptor, adaptor protein, BAR domain proteins, dynamin, SNARE proteins, Rab-GTP, Rab effector. Describe the role of phosphoinositides, listing the compartment specificity of various PIPs.

Answer 1

In addition to protein processing, the ER and Golgi also take care of some types of protein transport. Vesicles (membrane-bound bubbles, essentially) pinch off from the ER, Golgi, and other membranous organelles, carrying with them whatever soluble molecules were inside the fluid that was enclosed as well as any molecules embedded in that section of membrane. These vesicles then catch a ride on a molecular motor such as kinesin or myosin, and travel along the cytoskeleton until they dock at the appropriate destination and fuse with the target membrane or organelle. In general, vesicles move from the ER to the cis Golgi, from the cis to the medial Golgi, from the medial to the trans Golgi, and from the trans Golgi to the plasma membrane or other compartments. Although most movement is in this direction, there are also vesicles that move back from the Golgi to the ER, carrying proteins that were supposed to stay in the ER (e.g. PDI) and were accidentally scooped up within a vesicle.

The major types of coat proteins used in vesicle formation are COPII, COPI, and clathrin.COPII coat proteins form the vesicles that move from ER to Golgi. COPI coat proteins are used between parts of the Golgi apparatus as well as to form vesicles going from the Golgi back to the ER. Finally, clathrin is used to form vesicles leaving the Golgi for the plasma membrane as well as for vesicles formed from the plasma membrane for endocytosis.....The coat proteins come off shortly after vesicular release. For clathrin, the process involves Hsc70, an ATPase. However, for COPI or COPII coated vesicles, hydrolysis of the GTP on ARF/Sar1p appears to weaken the coat protein affinity for the adapters and initiates uncoating. The GTPase activator is ARF GAP (or Sec23p) and is an integral part of the COP I (or II) coat.

Clathrin is the best described of the three, and the vesicular coats are made from arrangements of clathrin triskelions . Each triskelion is composed of three heavy chains joined together at the C-terminus, and three light chains, one associated with each heavy chain. The heavy chains of different triskelions interact along the length of their heavy chain “legs” to create a very sturdy construct. The light chains are unnecessary for vesicle formation, and are thought to help prevent accidental interactions of clathrin molecules in the cytoplasm.

clathrin binds to adapter proteins which are bound to transmembrane cargo receptors, linking the membrane with the clathrin.....The ARF1 (or Sar1p) is used to recruit adapter proteins that bind to the “tail” end of membrane-bound receptor proteins. The business end of these receptors binds to car- go molecules that need to be packaged into the vesicle. The adapter proteins act as the link between the membrane (through the receptors) and the coat proteins. For clathrin, the adapter proteins are AP1 for trans-Golgi-derived vesicles and AP2 for endocytic vesicles.

Dynamin molecules are globular GTPases that contract upon hydrolysis of GTP. When they associate around the vesicle stalk, each dynamin protein contracts, with the combined effect of constricting the stalk enough that the membrane pinches together, sealing off and releasing the vesicle from the originating membrane.

This process is aided by tethering proteins which initially make contact with an incoming vesicle and draw it close enough to the target to test for SNARE protein interaction. Other proteins on the vesicle and target membranes then interact and if the SNAREs match, can help to “winch” the vesicle into the target membrane, whereupon the membranes fuse.....as a vesicle approaches the target membrane, the tethering protein Rab-GTP, which is linked to the target membrane via a double geranylgeranyl lipid tail, loosely associates with the vesicle and holds it in the vicinity of the target membrane to give the SNARES a chance to work.

Phosphoinositides are a family of minority acidic phospholipids in cell membranes. Their principal role is instructional: they interact with proteins. Each cellular membrane compartment uses a characteristic species of phosphoinositide. This signature phosphoinositide attracts a specific complement of functionally important, loosely attached peripheral proteins to that membrane. For example, the phosphatidylinositol 4,5-bisphosphate (PIP2) of the plasma membrane attracts phospholipase C, protein kinase C, proteins involved in membrane budding and fusion, proteins regulating the actin cytoskeleton, and others. Phosphoinositides also regulate the activity level of the integral membrane proteins. Many ion channels of the plasma membrane need the plasma-membrane-specific PIP2 to function. Their activity decreases when the abundance of this lipid falls, as for example after activation of phospholipase C. This behaviour is illustrated by the suppression of KCNQ K+ channel current by activation of M1 muscarinic receptors; KCNQ channels require PIP2 for their activity. In summary, phosphoinositides contribute to the selection of peripheral proteins for each membrane and regulate the activity of the integral proteins.