Secretion and Endocytosis

Learning Objectives

  • Diagram the pathway a protein takes from the ER to the plasma membrane.
  • Describe the role of coat proteins in incorporating secreted proteins into vesicles.
  • Describe the role of Rabs and SNAREs in mediating fusion between vesicles and specific organelles.
  • Define the role of glycosylation in targeting proteins to lysosomes.
  • List the potential fates of proteins endocytosed from the plasma membrane.

Lecture Content

Introduction

The secretory pathway in eukaryotic cells is used to send proteins and lipids to the plasma membrane and certain membrane-bound organelles and to release material outside the cell. There are two types of secretion: constitutive and regulated. Constitutive secretion is the default pathway and is used primarily to replenish material at the plasma membrane and certain membrane-bound organelles. Regulated secretion terminates in secretory vesicles that store secreted material until a signal triggers fusion with the plasma membrane. Both types of secretion use the same pathway but signal sequences divert proteins into the regulated pathway. Cells also retrieve material from the plasma membrane through endocytosis. This material can either be recycled to the plasma membrane or degraded in the lysosome.

Principles of the Secretory Pathway

Proteins and lipids are synthesized in the ER and then transported to the Golgi. Proteins are sorted in the Golgi and sent to the plasma membrane, lysosome or secretory vesicles. Transport of protein and lipid between membrane-bound compartments is mediated by vesicles that bud from one compartment and then fuse with the subsequent compartment. Rabs, tethers and SNAREs increase the probability that vesicles fuse with the correct target membrane. Cells maintain the integrity and functionality of the ER and Golgi by inhibiting resident proteins from entering vesicles and retrieving those proteins that do escape.

Glycosylation

Glycosylation is the covalent attachment of sugars to proteins that happens to most proteins in the ER. Glycosylation helps proteins fold, targets proteins to specific organelles (e.g. lysosome), and inhibits proteolysis. In addition, many proteins on the surface of cells and in the extracellular matrix that surrounds cells are heavily glycosylated for a variety of biological purposes.

N-linked glycosylation occurs in the ER and involves the addition of a group of 14 sugars to the amine group of asparagines. The groups contains a mix of N-acetylglucosamine, mannose and glucose. The glucose residues are removed in the ER before the protein is transported to Golgi. In the Golgi the sugar side chains can be further modified by removal and addition of different sugars.

O-linked glycosylation is the second form and involves the addition of sugars to serines or threonines. O-linked glycosylation likely starts in the Golgi by the addition of a single sugar. Other enzymes add sugars in groups of two and the sugar side chains can become extremely long.

The Golgi complex is a stack of membrane cisternae with unique biochemical compositions. The cisternae are usually termed cis, medial, trans and trans-Golgi network with protein entering the cis from the ER and exiting from the TGN. The cisternae appear to contain a unique set of enzymes that modify sugar side chains on proteins. For example, mannose is removed primarily in the medial cisterna whereas galactose is added in the trans cisterna.

Vesicular Transport

Transport between membrane compartments is mediated by small vesicles. The vesicles contain a protein coat that drives vesicle formation and recruits proteins into vesicles. Vesicles are targeted to the correct compartment by a combination of Rab proteins and SNAREs. Rabs are a large family of small GTP-binding proteins, and each membrane compartment in the secretory pathway appears to contain a unique Rab protein. SNAREs are proteins on vesicles and membrane compartments that pair to mediate fusion. SNAREs comprise another large family of proteins and different compartments likely contain unique SNARE proteins.

Vesicule Formation

Formation of vesicles from the ER is most clearly understood and will serve as an example of how vesicle form. The mechanism is likely similar for other compartments. Assembly of a protein coat drives formation and coat assembly starts with the binding of the small GTP-binding protein Sar1. Sar1-GTP associates with ER and inserts a small helix into the outer leaflet of the ER membrane bilayer to initiate curvature of the membrane. Sar1-GTP recruits two other sets of proteins that make up the vesicle coat: the Sec23-Sec24 complex that binds to cargo proteins and the Sec13-Sec31 complex that helps drive the formation of the vesicle. Cargo selection for most proteins requires a signal sequence that interacts with the Sec23-24 complex. Soluble proteins within the lumen of the ER associate with cargo receptors which contain a signal sequence that binds Sec23-Sec24. The coat complex that surrounds vesicles from the ER is called COP II.

Targeting Vesicles to Correct Compartment

Two sets of proteins appear to help vesicles fuse with the correct target membrane. One set involves tethers that localize to target membrane compartments and interact with components of the vesicle coat. Several different tethers have been identified in cells and each appears to localize to a distinct compartment. Tethers form structures that extend away from the compartment membrane into the cytosol. This may help tethers interact with vesicles arriving from the previous membrane compartment.

A second set of proteins that helps correctly target vesicle to the appropriate membrane is the SNAREs. SNAREs also mediate fusion between membranes. Vesicles contain one SNARE protein (vSNARE) and membrane compartments contain 2 to 3 SNARE proteins (tSNAREs). SNARE proteins on vesicles and membrane compartments interact with specificity. Animal cells express 35 different SNARE proteins but only certain sets of SNAREs interact with each other. By localizing those SNAREs which interact only to vesicles and their target membrane, cells ensure that vesicles fuse to their correct target membrane.

Membrane Fusion

SNARE proteins mediate fusion between vesicles and their target membrane compartment. SNARE proteins contain long regions that form helical structures. The helical domains in vSNAREs and tSNAREs interact and appear to zipper up. The energy released through complete pairing of vSNAREs and tSNAREs is thought to drive fusion between vesicle membrane and compartment membrane, though the exact mechanism remains unclear.

Some vesicles dock on their target membrane but do not fuse. For example, secretory vesicles store proteins and other small molecules until the cell is signaled to release them. Some secretory vesicles dock on the plasma membrane through interaction of vSNAREs and tSNAREs, but the SNAREs are prevented from completely pairing to drive membrane fusion. External signals trigger the removal of the pairing inhibition, allowing the vesicles to fuse with the plasma membrane.

Protein Sorting in trans-Golgi Network

Upon reaching the trans-Golgi network, most proteins are targeted to their final destination. The default pathway appears to be transport to the plasma membrane, as the plasma membrane needs to continuously replace lipids and proteins. Other proteins are sorted to lysosomes and secretory vesicles. The signal to send a protein to the lysosome involves the sugar side chain. Most lysosomal proteins contain mannose 6-phosphate which is added in the cis-Golgi. The receptor that binds mannose 6-phosphate resides in the trans-Golgi network and recruits coat proteins to the trans-Golgi network. Clathrin forms the coat around these vesicles, and the vesicles accumulate lysosomal proteins before budding from the trans-Golgi network. These vesicles fuse with endosomes. The lumen of endosomes has a low pH causing the mannose 6-phosphate receptor to dissociate from lysosomal proteins. The mannose 6-phosphate receptor is returned to the trans-Golgi network and the vesicle containing the lysosomal proteins matures into a functional lysosome.

Some proteins are sorted into secretory vesicles that store these proteins until the cell is signaled to release them. The mechanism of by which proteins are sorted into secretory vesicles as these proteins do not share a common sorting signal sequence.

Endocytosis

Cells not only release material to the external environment but they also take up material from outside the plasma membrane through endocytosis. There are several forms of endocytosis.

Phagocytosis allows some cells (macrophages, neutrophils) to engulf and take up large particles such as microorganisms and dead cells. Phagocytosis involves the protrusion of the plasma membrane around the particle. Protrusion is driven by actin polymerization. The plasma membrane eventually surrounds the particle and fuses to completely enclose it and form a large endocytic vesicle.

Pinocytosis forms much smaller vesicles (~ 100 nm) and allows cells to take up small amounts of extracellular fluid and portions of the plasma membrane. One form of pinocytosis is clathrin-mediated endocytosis that allows cells to take up specific proteins from the cell surface.

Clathrin-mediated endocytosis begins with the formation of pit in the plasma membrane. The pit is surrounded on the cytoplasmic side by adaptor proteins that link clathrin to the pit. The adaptors also interact with proteins in the plasma membrane that are targeted for endocytosis. The pit can accommodate ~ 1000 proteins. Polymerization of clathrin drives formation of a vesicle that eventually pinches off from the plasma membrane. The GTPase dynamin catalyzes the pinching off reaction. The clathrin-coated vesicles fuse with endosomes where the low pH dissociates ligands from receptors. Some proteins are then returned to the plasma membrane whereas others are targeted to the lysosome where they are degraded.