Vesicles and Secretion

What is the secretory pathway?

Digestive enzymes are secreted into the pancreatic duct, whilst hormones are secreted into the blood. Insulin receptors have to be present in the plasma membrane. Transport of proteins and lipids to the plasma membrane occurs in secretory vesicles, and secretion can be either constitutive or regulated. Exocytosis means fusion of secretory vesicles.

We can study the secretory pathway in a few different ways: pulse-chase experiments, fluorescence live imaging or genetic analysis of yeast mutants.

Pulse-chase experiments

Pulse-chase experiments means incubation with labelled amino acids for a short period, a pulse, and analysing cells after different time intervals, a chase.

Fluorescence live imaging

Create a DNA construct which encodes the protein that you’re interested in + encoding a fluorescent protein, beefore transferring the DNA construct into cells → this allows for imaging in living cells!

Genetic analysis of yeast mutants

Turn to forward genetics, generate random mutations and screen for mutants with defects in budding (yeast, requires new membrane).

How do vesicles form?

Vesicle formation requires coating of the membrane, and vesicles on different routes have different coats. There are three main vesicle types:

• COPI (COP = Coat Protein)

• COPII

• Clathrin

The coats are polymers, and polymerization of the coat proteins cause bending of the membrane, which can regulate which cargo proteins are in the vesicle or in the vesicle membrane.

How do COPII vesicles assemble?

COPII vesicles move from ER to cis-Golgi. Sec12 is located in the ER membrane, and acts as a GEF for Sar1. Sar1 activation causes a change in its conformation, which allows binding to the ER membrane. Sar1-GTP binds COPII proteins to assemble the coat. After relase of the vesicle, Sar1 hydrolyzses GTP and the coat disassembles. Sec12 is ONly present in the ER, and COPII vesicles only assemble at the ER for transport to the cis-Golgi. The last step includes pinching off the vesicle. This requires GTP hydrolysis of Sar1-GTP at the connection to the vesicle, the “neck”. Other small GTPases function in the assembly of the coats of other types of vesicles.

How are proteins sorted into vesicles?

Proteins can interact with specific coat proteins via specific amino acid sequences, known as sorting signals. Soluble proteins, e.g. in the ER lumen, bind indirectly to coat proteins by their sorting signals binding to membrane cargo recepters that again bind to the coat protein. Many membranes proteins transported by COPII vesicles have a di-acidic sorting signal that binds to Sec25.

What is retrograde transport?

COPII vesicles will accidentally transport some proteins to the cis-Golgi that function in the ER, and COPI will transport these back to the ER during retrograde transport.

Rab-proteins

Rab proteins are a family of small G proteins, and Rab-GDP binds to vesicles via a lipid molecule attached to the protein. A GEF in the vesicle membrane exchanges GTP for GDP. The activated Rab (Rab-GTP) can bind to Rab effector proteins on the target membrane or on motor proteins.

Snare complex

Additionally, vesicles have specific v-SNARE proteins on their surface, which binds to t-SNARE proteins on the target membrane to form a SNARE complex. v-SNARE and t-SNAREs form a stable bundle of four alpha helices that bring vesicle and target membrane close together, which allows for the membranes to fuse.

Disassembly of the SNARE complexes requires energy, which is provided by ATP hydrolysis by NSF.

Transport through the Golgi

Carbohydrate modification of proteins start in the ER, whilst further modifications are added in the Golgi. Different Golgi compartments contain different glycosyltransferases and glycosidases. Retrograde COPI vesicle transport leads to maturation of Golgi compartments.