Introduction to the Secretory Pathway: Part 1 Lecture Notes

Key Words and Terms

electron microscopy, vesicles, vesicle docking, membrane fusion, plasma membrane, endoplasmic reticulum (ER), Golgi apparatus, temperature sensitive (ts) mutations

Lecture Notes

The secretory pathway is used by all cells to deliver molecules such as proteins and lipids to different parts of the cell and outside the cell. It is involved in everything from cell surface growth to the construction of organelles. The mechanisms of this process are conserved among all organisms, particularly eukaryotes.

The secretory pathway in human nerve cells

The secretory pathway transmits neurotransmitters between nerve cells.

A thin section of a nerve cell can be visualized by electron microscopy.

The synapse is the clear area between adjacent membranes of adjoining cells.

Vesicles are the basic unit of transmission in the brain and of protein secretion in all cells. They are composed of a membrane bilayer very much like the bilayer at the cell surface and a clear interior that contains the chemicals or macromolecules that are to be secreted.

In the resting state (no stimulus) vesicles are produced and delivered to the plasma membrane where they dock but do not merge.

In response to a stimulus, vesicle membranes merge with the plasma membrane in a process called membrane fusion. As the two membranes become continuous, the contents of the vesicle come into physical contact with the outside of the cell.

All vesicle contents are secreted, but the membrane portion of the vesicles is internalized and recycled for use in future vesicle formation.

Freeze-fracture electron microscopy shows the surface topology of nerve cells.

In the resting state the membrane looks smooth. The small particles represent integral membrane proteins at the cell surface.

The membrane fusion and synaptic discharge that occur in response to a stimulus are observed as “holes” or “dimples” in the membrane. These holes represent the interior content of vesicles as they deliver neurotransmitters into the synapse.

The secretory pathway in baker’s yeast (Saccharomyces cerevisiae)

The secretory pathway delivers newly synthesized macromolecules to the cell surface.

Yeast cells divide by budding. Bud enlargement requires a significant increase in the area of the bud surface. Electron microscopy helps reveal the process needed for this to occur.

The plasma membrane of yeast is surrounded by a cell wall composed of chitin and various other polysaccharides that confers rigidity. It also differentiates yeast cells from mammalian cells.

Membranes inside the yeast cell are very similar to those found in animal cells.

Nucleus – yeast cells are eukaryotic.

Vacuole – a digestive organelle, similar to the lysosome in animal cells.

Endoplasmic reticulum (ER) – this membrane is characteristic of the secretory pathway and is involved in biosynthesis of macromolecules, some of which eventually leave the cell.

Golgi apparatus – also characteristic of the secretory pathway, this is the “bus station” of the cell. It receives material from the ER and sifts molecules according to their final destination.

Small vesicles – seen in rapidly growing cells, these are very similar to the vesicles required for neurotransmitter secretion but are instead responsible for expanding the bud surface by discharging molecules that will become part of the plasma membrane or cell wall.

Genetic dissection of the yeast secretory pathway

Genetic techniques are more easily applied to yeast than human cells.

The hypothesis: if one were to interrupt the flow of vesicles by interfering with their production, targeting, docking, or fusion, vesicles or precursor membranes should build up inside the cell at the expense of cell surface expansion.

But how do we study a process that is essential for cell viability when deleting an essential gene involved in this process should cause cells to die?!?

One approach is to create temperature sensitive (ts) mutations that selectively interfere with a protein’s function at high but not low temperatures. Such mutations affect proteins in such a way that they fold properly at low temperatures but become unstable (unfold) at higher temperatures.

In 1979 Peter Novick identified a ts sec1 mutant that could grow at room temperature (25°C) but not at body temperature (37°C).

sec1 mutants incubated at 37°C arrest because vesicles, no longer restricted to the growing bud, accumulate to concentrations many fold higher than typical and fill up the entire cytoplasmic volume.

Electron micrographs show that these vesicles carry membrane proteins and other cargo but are unable to merge with the plasma membrane.

sec1 cells arrested at 37°C can resume growth when the temperature is shifted back down to 25°C, but cells grown too long at 37°C eventually die.

Because sec1 mutants have an increased buoyant density at 37°C due to an accumulation of mass without an increase in cell volume, similar mutants were identified using a density gradient.

At least 10 different genes have been identified that are required at the same step in this secretory pathway as SEC1 – the docking and fusing of vesicles at the plasma membrane.

Comparing the sequence of these proteins to mammalian proteins led to the identification of mammalian homologs. The docking and fusing of vesicles at the plasma membrane is a fundamentally conserved process.

Additional secretory mutant phenotypes

Not all secretory mutants show the sec1 vesicle accumulation phenotype.

sec7 ts mutants have a larger, elaborated Golgi apparatus when grown at 37°C.

Some essential function of Sec7p permits proteins to move out of the Golgi and into secretory vesicles.

sec7 sec1 ts double mutants show the sec7 phenotype rather than the sec1 phenotype at 37°C. Therefore, the SEC7 process precedes the SEC1 process.

Mutations in over 30 different genes produce an exaggerated ER with elaborated tubules and an enlarged lumen.

The ER in these mutants becomes engorged with molecules that cannot be passed along the secretory pathway.

Double mutant analysis of these mutants with sec7 mutants reveals that this station precedes the Golgi station.

Overview of the yeast secretory pathway

1. Secretory proteins enter the ER as they are synthesized by ribosomes.
2. These proteins are packaged into vesicles that bud from the ER and are transported to the Golgi apparatus. There is a bidirectional flow of materials between the ER and the Golgi (see Schekman lecture part 2).
3. At the Golgi material is sorted.
   1. Some material is diverted to the vacuole.
   2. Other molecules are packaged into vesicles and delivered to the plasma membrane.