Protein Processing III: Secretion, Lysosomal and Endocytotic Pathways.

Summary of Protein processing are Post-Golgi stages

A. The Secretory Pathway.

Overview: There are two different secretory pathways, the Regulated Pathway and the Constitutive Pathway. In the regulated pathway proteins are consolidated into vesicles that are stored in the cell until they are secreted in response to a specific signal. In the constitutive pathway vesicles continuously form and carry proteins fron the Golgi to the cell surface.

1. Proteins are modified and sorted in the Golgi.

The flow of material in the Golgi apparatus is from cis to trans Figure 14-24 .

As proteins proceed through the Golgi they are sorted and processed.

The trans Golgi network is a major sorting centre. Vesicles leave the trans Golgi network for a number of destinations. These include:

incorporation into dense core secretory vesicles that are released through the regulated secretory pathway in response to a specific stimulus, and
vesicles containing membrane and proteins that are released continuously to the surface via the constitutive secretory pathway,
inclusion in lysosomes (for example enzymes involved in intracellular digestion) via the lysosomal pathway.

How are proteins targeted to these three different fates? - The fates of the proteins are thought to be determined by features of the proteins themselves.

Proteins destined for the regulated secretory pathway form aggregates in the trans Golgi network,
while those destined for the constitutive secretory pathway do not.

Protein aggregates containing proteins destined for regulated secretion begin to develop in the trans Golgi network.

The aggregation occurs in response to the acidic conditions of the trans Golgi network. Aggregation does not occur under the pH neutral conditions of the earlier parts of the secretory pathway.

These protein aggregates give rise to the 'dense core secretory granules' that are the typical storage vesicles of the regulated secretory pathway.

Fig. 14-26. Insulin containing dense core secretory in a islet beta cell of the pancreas. One of the granules in being exocytosed.

The regulated secretory pathway is used for proteins that are stored and secreted on demand. For example,

insulin is produced by the beta cells of the pancreas and stored in dense core secretory granules. When blood sugar increases to a threshold level, insulin containing secretory granules fuse with the plasma membrane, releasing insulin to the blood, as shown above.
The digestive enzymes produced by the acinar cells of the pancreas are stored in dense core granules called zymogen granules and are released into the duct leading to the gut following feeding.

Proteolytic clevage of many secreted proteins occurs during the maturation of dense core secretory granules.

The regulated exocytosis pathway operates in cells specialized for secretion and produces cell product on demand. Dense core secretory granules dock at the cell surface and release their contents when they are stimulated to do so. This usually occurs through the dramatic rise in intracellular Ca⁺⁺ concentration that brought about by a calcium action potential.

For example, in nerve endings synaptic vesicles containing neurotransmitters (e.g. acetylcholine) fuse with the plasma membrane and release their contents when voltage activated calcium channels open in response to depolarization of the membrane.

The constitutive exocytosis pathway operates continually in all cells and supplies a continuous stream of vesicles containing lipids and proteins for the plasma membrane. This includes the glycoproteins form a major part of the extracellular matrix.

The last ten pages of the chapter (pp. 467-77) give examples of operation of the secretory pathway. You are to study these on your own.

Our expectation is that you will learn the processes in detail using at least one of the following examples:

secretion of cell wall polysaccharides
secretion of collagen in animal cells
secretion of a glycoprotein.

B. The Lysosomal Pathway

Overview: In the lysosomal pathway proteins destined for lysosomes (e.g. digestive enzymes) contain a special targeting tag, mannose 6 phosphate, and are sequestered by mannose 6 phosphate receptors into vesivles bound for late endosomes. Here the lysosomal proteins are separated from their receptors and are sent by vesicles to lysosomes (red arrows) and the receptors are returned to the Golgi (blue arrow).

The lysosomal pathway directs proteins to lysosomes.

Lysosomes are vesicles that contain digestive enzymes and that are the site of intracellular digestion. Proteins destined for lysosomes have a special targeting signal.

They are all glycosylated and some of the mannose residues in the attached oligosaccharides are phosphorylated to form mannose-6-phosphate.
Mannose-6-phosphate is the targeting signal for lysosomal proteins.

The trans Golgi network contains a special mannose-6-phosphate receptor that binds to and results in the concentration of proteins carrying oligosaccharides bearing mannose-6-phosphate into vesicles that are destined for lysosomes.

C. Endocytic Pathway

Overview: in the endocytotic pathway, external proteins bind to receptors in the plasma membrane and are incorporated into vesicles that are carried to early endosomes (green arrow), where the receptors are separated and returned to the cell surface (blue arrow). Proteins from early endosomes is then moved to late endosomes and then to lysosomes (green arrows) where the proteins are digested by the lysosomal enzymes.

We are dealing with two related phenomena:

Phagocytosis - the ingestion of large particles by cells. The particles are taken into phagosomes or food vacuoles. This occurs primarily in specialized cells, e.g. macrophages, or protists such as Amoeba or Paramecium.
Pinocytosis - the ingestion of fluid and macromolecules via small vesicles < 150 nm in diameter. This is carried out by all cells.

In phagocytosis bacteria or other large particles are taken into membrane vesicles. Phagocytosis occurs in response to activation of surface receptors. This results in the opening of calcium channels, and the increase in the internal Ca⁺⁺ concentration triggers the formation of a phagosome or food vacuole. Phagocytic cells play an important role in removal of foreign organisms (bacteria) and in the scavenging of dead or damaged cells.

Figure 14-28. Macrophage engulfing two red blood cells. Notice the collar-like pseudopodium creeping over the surface of the red blood cells (red arrows). Given what you know about the cytoskeleton, what do you think is happening inside the macrophage to make this happen?

The book says that macrophages eat 10¹¹ red blood cells in each of us each day!!! That is a lot of cells! This occurs in the spleen.

Pinocytosis. This involves the uptake of macromolecules and fluid by cells.

The macromolecules bind to the cell surface. This is followed by the formation of a vesicle containing the surface membrane with the attached molecules and the surrounded fluid.

Macrophages, for example, ingest about 25% of their own volume in fluid each hour and an area of membrane equal to its surface every 30 minutes.

Pinocytosis takes place via the formation of clathrin coated pits and vesicles. The membrane vesicles eventually fuse with endosomes, and the macromolecular material they contain is transfered to lysosomes where it is digested.

The binding of macromolecules to proteins at the cell surface triggers a process known as receptor mediated endocytosis.

The concentration of bound molecules triggers the formation of a vesicle. Macromolecules may be concentrated up to 1000-fold by accumulation on the cell surface before incorporation into vesicles. The concentration of cargo proteins occurs through the process discussed earlier in the lecture on vesicle formation:

Cargo molecules bind to receptor molecules.
The receptors then bind to adaptin and coat protein (clathrin, in this case) that result in their concentration in the area of the forming membrane bud that will give rise to the endocytotic vesicle.

Endocytosed macromolecules are sorted in Early Endosomes. Early endosomes are reltively small vesicles contained within the cytosolic compartment.

Early endosomes have an acidic internal environment due to proton pumps in their membrane.
The acidic environment causes many receptors or carrier proteins to release their cargo of bound macromolecules.
Most of the receptors are sorted out and returned to the plasma membrane by vesicle transport. Macromolecules that are removed from their receptors are transferred to lysosomes for digestion.

In a well studied case with LDL receptors (see below) a receptor makes one round trip into the cell and back every 10 minutes.

Fig. 14-29. Click to enlarge.

Receptor-mediated endocytosis of Low Density Lipoprotein (LDL).

The protein binds to receptors on the cell surface and in internalized in clathrin-coated vesicles.
The vesicles then lose their coats and fuse with endosomes.
In the acidic environment of the endosome, LDL separates from its receptor.
The LDL is then transferred to a lysosome, where it is degraded to release free cholesterol
The LDL receptors are returned to the plasma membrane via transport vesicles.

Early endosomes are main sorting sites on the endocytotic pathway, as the trans Golgi network serves this function in the secretory pathway.

Endosomes receive a number of different types of receptors along with their cargo.

Most of the receptors are returned to the same plasma membrane domain from which they came,
others are passed to lysosomes and are destroyed,
while others, with their cargo are transferred to another plasma membrane domain and eventually transfer their cargo from one extracelular space to another, e.g. from the distal to the lateral face of an epithelial cell. See Fig. 14-30

Proteins destined for digestion are carried by vesicles from early endosomes to larger vesicles called Late Endosomes. Late endosomes are also involved in receptor sorting and recycling, but these are the mannose-6-phosphate receptors that carry lysosomal proteins from the trans Golgi network to late endosomes. Late endosomes receive vesicles from the trans Golgi network that contain lysosomal enzymes bound to mannose-6-phosphate receptors as described above.

In late endosomes the mannose-6-phosphate receceptors and their lysosomal enzyme cargo separtate due to the acidic pH of the endosome.

The receptors are then concentrated into vesicles and returned to the trans Golgi network.

The lysosomal enzymes and the macromolecules to be digested that came in from early endosomes are transferred to lysosomes.

These processes are summarized in the following diagram:

Recycling of cell surface receptors and mannose- 6 - phosphate receptors by endosomes.

The green circles represent cargo molecules coming from formation of endocytotic vesicles at the cell surface.
The red squares represent lysosomal enzymes carried from the trans Golgi network to the endosome by mannose-6 -phosphate receptors.
Both types of receptors are recycled.
Transport vesicles carry both lysosomal enzymes and macromolecules to be degraded to lysosomes.

Sorting processes in cells are not perfect.

For example, some lysosomal proteins end up in vesicles that are secreted. Cells have a retrieval pathway to catch these errant lysosomal enzymes.

The lysosomal enzymes bind to mannose- 6- phosphate receptors at the plasma membrane,
the proteins are brought to endosomes and sent on to lysosomes while
the receptors are returned to the trans-Golgi network.

This rather elaborate system is simply a consequence of the normal operation of the sorting pathways. The mannose-6-phosphate receptors are at the surface because the targeting process that brings most of them back to the trans Golgi network is also not perfect.

Lysosomes are the digestive system of cells.

These vesicles are a specialized set of secretory vesicles produced by the Golgi and contain a mixture of some 40 types of digestive enzymes, including those that degrade nucleic acids, proteins and lipids.
All of these enzymes have optimum activity at about pH 5.
This acid pH is maintained in lysosomes, as in endosomes, by proton pumps in the membrane.
The membrane of the lysosome is resistant to action of its own digestive enzymes due to the extensive glycosylation of the proteins on the lumen side of the membrane.