Secretion - Exocytosis

What is constitutive vs regulated secretion?

constitutive secretion is always happening; secretory vesicles form and immediately fuse with the plasma membrane to release their contents; regulated secretion is controlled; secretory vesicles form but they don’t immediately fuse with the plasma membrane, rather they sit beneath the plasma membrane until some sort of signal causes them to fuse with the membrane and release their contents (ex. neurotransmitters)

What technique helped make exocytosis visible?

Total Internal Reflection Microscopy (TIRF); in this technique, the light is refracted at an angle at which it is all reflected, but some light energy penetrates the sample above, creating an evanescent field; this energy activates any GFP within 100 nm of the slide (so close to the surface of the cell), so anything being secreted from the cell can be seen

Do cargo proteins float around in the middle of a vesicle?

No, they are bound to the lumenal domain of a receptor protein, so the middle of a vesicle is mostly empty with all the proteins being on the edge

Do secretory vesicles fuse with just any part of the membrane?

No, where they fuse with the membrane is regulated by many different mechanisms, like in the apical and basolateral domains of intestinal epithelial cells

How do proteins get sorted to the basolateral domain of intestinal epithelial cells?

A dileucine motif (LL) in a protein is a signal for a vesicle to go from the TGN to the basolateral domain

How do proteins get sorted to the apical domain of intestinal epithelial cells?

There is no specific sorting sequence, rather it is based on the thickness of the membrane in the compartment; there is a gradient of membrane thickness through the secretory pathway, with membrane thickness increasing (from 5 nm to 8 nm) as you move outward from the ER; the apical domain is thicker than the basolateral domain because it has many sphingolipids with long tails; this means that the vesicle coming from the TGN must also have a thick membrane; transmembrane proteins must have a long transmembrane domain in order to move into the thicker region of the TGN destined for the apical domain, so this process of exclusion makes sure only the proper proteins make it to the apical domain

Does all trafficking to the plasma membrane occur with the use of vesicles?

No, there is evidence for non-vesicular trafficking of lipids to the plasma membrane from the ER

How did the experiment with cholesterol and BFA point to non-vesicular trafficking?

In samples where BFA (a protein that disrupts ER → Golgi trafficking) was added, the percent of trafficked proteins to the plasma membrane decreased significantly, however the percent of trafficked cholesterol in the plasma membrane decreased only slightly, indicating that the cholesterol was getting to the plasma membrane via another mechanism, leading to the discovery of membrane-contact sites

What are membrane-contact sites?

They are sites of non-vesicular trafficking to the plasma membrane; at these sites, the ER and the plasma membrane come within nanometers of each other and there is a bulk transfer or lipids from the ER to the plasma membrane; this is facilitated by proteins that bridge the gap between the ER and the plasma membrane; these proteins have hydrophobic interiors that allow lipids to “slide” in bulk from the ER to the plasma membrane

In an intestinal epithelial cell, where does a protein with a short transmembrane domain get trafficked to (assuming it has no LL motif)?

It would remain in the Golgi because it cannot continue further along the secretory pathway since membrane thickness increases

What is the relative pH of each compartment in the secretory pathway and how does it relate to function?

• ER → 7.0, allows KDEL protein to unbind from KDEL receptor

• Golgi → ~6.0 - 5.7, KDEL protein is able to bind to KDEL receptor and mannose-6-phosphate can bind to M6P receptor

• Late endosome → 5.5, allows mannose-6-phosphate to unbind from M6P receptor; keeps acid hydrolases from fully functioning

• Lysosome → 5.0, allows acid hydrolases to become fully functional