Lecture 6 - endomembrane system Flashcards
What are the organelles of the endomembrane system?
ER
ER derived organelles - nucleus and peroxisomes and lipid bodies
golgi
endosomes
lysosomes/vacuoles
secretory granules
plasma membrane
How are large amounts of material trafficked between organelle structure?
membrane bound transport vesicles
What are the four steps to trafficking through the endomembrane system via transport vesicles?
- cargo containing vesicle buds off donor membrane compartment: vesicle coat proteins select which donor membrane and soluble cargo proteins enter or not, nascent transport vesicle and regulate vesicle formation and budding
- 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
- vesicle fuses with proper acceptor membrane compartment, receptor proteins also regulate vesicle acceptor membrane fusion, vesicle donor membrane and lumenal cargo proteins incorporated into acceptor compartment
- 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 compartment
what do motor proteins do?
motor proteins direct vesicle movement by linking to vesicle surface and cytoskeleton element
What is the biosynthetic pathway?
materials transported from ER to golgi, endosomes and then lysosomes
What are two types of secretion?
- constitutive secretion
- regulated secretion
What is constitutive secretion?
- materials continually transported from golgi to pm and or released via exocytosis outside of cell in secretory vesicle (vesicle membrane components incorporated into pm)
What is exocytosis?
vesicle trafficking to and fusion with pm and release of contents
What is regulated secretion?
ER derived materials from golgi stored in secretory granules, in response to cellular signal, secretory granules fuse with pm and release (exocytosis) lumenal cargo into extracellular space, secretory granule membrane comp incorporated into pm
Does regulated secretion occur only in specialized cells?
yes
What are endocytic pathways?
- operate in opposite direction of secretory pathways (materials move into cell)
- materials from pm and/or extracellular space incorporated into cell via endocytosis and then transported to endosomes and lysosomes
What is endocytosis?
uptake of materials from pm and extracellular space into transport vesicles
does the amount of secretion vary between cell type?
yes
What are the secretions of each cell type?
yeast and plant cells - cell wall materials
pancreatic acinar cells - digestive enzymes
epithelial cells of small intestine - mucus
What cells are highly polarized?
pancreatic and intestine epithelial cells
How are the organelles organized within the cell?
basal end of cell - nucleus, rough ER
central region - golgi, lysosomes
apical end - secretory granules containing digestive enzymes
What is autoradiography and pulse chase radiolabeling experiments?
- demonstrated how proteins move through the secretory pathway - proteins associate with organelles and move via membrane bound itnermediates
What is the autoradiography and pulse chase radiolabeling experiment?
- pancreatic tissue briefly incubated with radioactive AA; labeled AA are incorporated into newly synthesized proteins
- tissue washed and incubated for varying lengths of time with non radioactive AA
- protein synthesis continues and radiolabeled proteins traffic through cell
- tissue gixed and exposed to x ray film
What are the results of audioradiography and pulse chase radiolabeling experiments?
brief chase (3 min) - rough ER (site of protein synth)
intermediate chase (20 mins) - golgi site of protein mod)
long chase (120 min) - secretory vesicles
What is live cell imaging using autofluorescent proteins?
using standard molecular bio techniques, gene encoding autofluorescent protein linked to gene of interest
recombinant gene fusion introduced into selected organism
intracellular localization and trafficking of actopically expressed fluorescent fusion protein visualized in living specimen using fluorescence microscopy
What are the steps to the modern pulse chase labeling like experiments to study protein transport through secretory pathways
temp sensitive viral glycoprotein (VSVG) fused to GFP and introduced into mammalian cultured cell
mutation in VSVG is reversible - allows for turning on and off intracellular transport
40 C (restrictive temp) - nascent VSVG protein misfolded and remains in ER due to quality control processes
32 C (permissive temp) - VSVG protein properly folds and transported from ER
What are the results from the VSVG GFP live cell fluorescence microscopy labeling exp?
40 C restrictive temp - rough ER
32 C permissive temp - golgi
What is subcellular fractionation using centrifugation?
- technique used to separate and purify specific organelles on the basis of their varying size and or densities
- allows for the study of organelles components structures and functions
What does homogenization generate?
homogenate
What does homogenization through centrifugation result in?
separates intact organelles of different size/density with increasing higher centrifugation speeds
What are microsomes?
fragments of ER membrane that fuse and reform into small, spherical vesicles
What are cell free systems approach aimed at characterizing the components and underlying molecular mechanisms of the endomembrane system?
characterization of the activities of specific endomembrane protein components in vitro - components purified from different organelle/ER microsomal fractions
isolated proteins incubated with liposomes
liposomes mixed with purified proteins - allows for study of protein in vitro membrane lipid environment
What are liposomes?
artificial spherical vesicles consisting of phospholipid bilayers surrounding aqueous center
What is the genetics approach aimed at characterizing the components and underlying molecular mechanisms of the endomembrane system?
mutant phenotype analysis
What is mutant phenotype analysis?
approaches to identify genes/proteins and steps in protein vesicle trafficking in endomembrane system by screening for mutant phenotypes
yeast - studied with conditional mutants
What are sec mutants?
collection of temp sensitive mutants that secrete proteins at permissive temp, but not at higher non permissive temp
What are the five major classes of sec mutants?
class A - accumulation of secretory proteins in cytosol
class B - accumulation in ER
class C
class D
class E
What do double sec mutants indicate?
order of steps in the pathway
