Lecture 6

Question 1

What is the end membrane system?

Answer 1

A dynamic coordinated and interconnected network of organelles and related structures.

*endoplasmic reticulum (ER)
*ER-derived organelles (nucleus & peroxisomes & lipid bodies)
*Golgi
*endosomes
*lysosomes/vacuoles
*secretory granules
*plasma membrane

Question 2

Trafficking through endomembrane system via transport vesicles
* involves four general steps

Answer 2

• Step 1: ‘cargo’-containing vesicle buds off ‘donor’ membrane compartment
  vesicle ‘coat’ proteins select which ‘donor’ membrane and soluble (lumenal) ‘cargo’ proteins enter (or not)
  nascent transport vesicle AND regulate vesicle formation and budding
• Step 2: nascent vesicle transported through cytoplasm to ‘acceptor’ membrane compartment
  vesicle receptor (‘coat’) proteins regulate intracellular trafficking of vesicle to proper acceptor membrane
  also involves molecular motors and cytoskeleton ‘highways’
• Step 3: vesicle ‘fuses’ with proper acceptor membrane compartment. receptors proteins also regulate vesicle-acceptor membrane fusion. vesicle (donor) membrane & lumenal cargo proteins incorporated into acceptor compartment
• Step 4: process of budding and fusion repeated and can occur in reverse direction
  other receptor proteins regulate recycling of proteins that ‘escape’ to acceptor membrane compartment back to donor membrane compartment

Question 3

What does transport involve in the biosynthetic pathway?

Answer 3

Biosynthetic pathway involves materials (e.g., lysosomal membrane and soluble proteins) transported from ER to Golgi, endosomes, and then lysosomes (vacuoles in plants)

or sometimes….

materials (e.g., HIV particles) transported, via exosomes, from endosomes to plasma membrane (pm) and extracellular space

Question 4

What are the two types of secretion in the biosynthetic pathway?

Answer 4

1. Consecutive secretion
2. Regulated secretion.

Question 5

What is sub cellular fractionation?

Answer 5

isolation of organelles by centrifugation

• homogenization - cell/tissue disrupted by gentle homogenization (ensure organelles remain intact)
• homogenate filtered (removes unbroken cells and large fragments) and subjected to differential centrifugation
  separates intact organelles/cellular components of different size/density with increasing higher centrifugation speeds

Question 6

What are microsomes ?

Answer 6

Fragments of ER membrane (and/or plasma membrane) that fuse and reform into small, spherical vesicles

Question 7

What is the role of equilibrium density gradient centrifugation?

Answer 7

• separates intact organelles/cellular components on basis of density
• organelle fraction layered on top of sucrose gradient (increasing density from top to bottom)
• centrifugation: individual organelles migrate to migrate to corresponding equilibrium densities
  -different layers of gradient removed and purified organelle fractions identified by EM and/or organelle marker proteins/enzymes
• determine composition of isolated organelles using proteo/lipidomics and/or use in cell-free (in vitro) import and vesicle trafficking assays

Question 8

Cell free systems

Answer 8

Cell free systems

• characterization of the activities (i.e., functions) of specific endomembrane protein components in vitro – components purified from different organelle/ER microsomal fractions
• isolated proteins incubated with liposomes - artificial, spherical vesicles consisting of phospholipid bilayer surrounding aqueous center
• liposomes mixed with purified proteins – allows for study of protein(s) in vitro in ‘natural’
  membrane lipid environment
  e.g., protein(s) involved in formation of transport vesicles cause liposome membrane ‘budding’
• allows for processes underlying protein/vesicle trafficking in endomembrane system to be reconstituted in vitro

Question 9

What is an example of an approach aimed at categorizing the components and underlying molecular mechanisms of the endomembrane system?

Answer 9

Cell free systems -biochemistry approach
Mutant phenotype analysis -genetic approach

Question 10

Mutant phenotype analysis

Answer 10

• approaches to identify genes/proteins and steps in protein/vesicle trafficking in
  endomembrane system by screening for mutant phenotypes
• vesicle transport,/endomembrane organization evolutionarily conserved * yeast - secretory pathway essential – studied with conditional mutants
• secretory (‘sec’) mutants – collection of temperature-sensitive mutants that secrete proteins at permissive temperature, but not at higher nonpermissive temperature