Endomembrane System Part 1 - Intro and Methods of Analysis

Question 1

what do transport vesicles do

Answer 1

exchange large amounts of material between each organelle/structure

Question 2

what do vesicle coat proteins do

Answer 2

select which donor membrane and soluble cargo proteins enter nascent transport vesicle
regulate vesicle formation and budding

Question 3

what occurs in the biosynthetic pathway

Answer 3

materials made in ER are transported from ER to Golgi, endoscopes and then lysosomes

Question 4

what are the 2 types of secretion

Answer 4

constitutive
regulated

Question 5

what occurs in constitutive secretion

Answer 5

materials continually transported from Golgi to plasma membrane and/or released (via exocytosis) outside of cell in secretory vesicle

Question 6

exocytosis

Answer 6

vesicle trafficking to and fusion with pm and release of contents

Question 7

what are secretory vesicle membrane components incorporated into

Answer 7

pm - lumenal cargo proteins released into extracellular space

Question 8

where does regulated secretion occur

Answer 8

only specialized cells

Question 9

what is stored in secretory granules

Answer 9

ER-derived materials from Golgi

Question 10

what do secretory granules do in response to cellular signal

Answer 10

fuse with pm and release lumenal cargo into extracellular space

Question 11

what do secretory granule membrane components get incorporated into

Answer 11

pm

Question 12

what pathway operates in the opposite direction of secretory pathways

Answer 12

endocytic pathway - materials move INTO cell

Question 13

endocytosis

Answer 13

uptake of materials from pm and extracellular space into transport vesicles

Question 14

in the endocytic pathway, where do materials get transported to

Answer 14

endosomes and lysosomes

Question 15

what did autoradiography and pulse-chase radiolabeling experiments demonstrate

Answer 15

how proteins move through secretory pathway: proteins associate with organelle and move via membrane bound intermediates

Question 16

what did the results of autoradiography and pulse-chase radiolabeling define

Answer 16

secretory pathway and organization and coordination of protein trafficking in endomembrane system

Question 17

results of brief chase (3 min)

Answer 17

rough ER (site of protein synthesis)

Question 18

results of intermediate chase (20 min)

Answer 18

Golgi (site of protein modification)

Question 19

results of long chase (120 min)

Answer 19

secretory vesicles (including those fused with pm)

Question 20

how did recombinant gene fusion introduce into selected organism/cell

Answer 20

via cloning

Question 21

how does live-cell imaging using autofluorescent proteins work

Answer 21

using standard molecular bio techniques, gene encoding autofluorescent protein linked to gene of interest

Question 22

live-cell imaging using autofluorescent protein result

Answer 22

intracellular localization and trafficking of ectopically expressed fluorescent protein visualized in living specimen using fluorescence microscopy

Question 23

what is subcellular fractionation

Answer 23

isolation of organelle by centrifugation - size/density

Question 24

what is homogenization

Answer 24

cell/tissue disrupted by gentle homogenization - ensures organelles remain intact

Question 25

what is homogenate

Answer 25

filtered and subjected to differential centrifugation

Question 26

what does homogenate remove

Answer 26

unbroken cells and large fragments

Question 27

what does differential centrifugation separate

Answer 27

intact organelles/cellular components of different size/density with increasing centrifugation speeds

Question 28

2 components of differential centrifugation

Answer 28

pellet at bottom supernatant

Question 29

what is supernatant

Answer 29

liquid at the top of the centrifuge tube

Question 30

result of differential centrifuge in pellet of 600g after 10 min

Answer 30

nuclei is isolated in pellet

Question 31

once the nuclei is isolated in the pellet, what is the supernatant subject to

Answer 31

15Kg x 5 min - mix of mitochondria, lysosomes etc 100Kg x 60 min - pm and ER microsomes

Question 32

what are microsomes

Answer 32

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

Question 33

what can individual organelles in each pellet fraction endure

Answer 33

can be further purified

Question 34

what does equilibrium density gradient centrifugation separate

Answer 34

intact organelles/cellular components on the basis of density

Question 35

cell-free system

Answer 35

characterization of the activities of specific end-membrane protein components in vitro

Question 36

liposomes

Answer 36

artificial, spherical vesicles consisting of phospholipid bilayer surrounding aqueous centre

Question 37

what do liposomes get mixed with in cell-free systems

Answer 37

purified proteins

Question 38

what does the mix of liposomes and purified proteins in cell-free systems allow for

Answer 38

study of proteins in vitro in natural membrane lipid environment

Question 39

what do cell-free systems allow for

Answer 39

processes underlying protein/vesicle trafficking in end-membrane system to be reconstituted in vitro

Question 40

what does mutant phenotype analysis approach to identify

Answer 40

genes/proteins and steps in protein/vesicle trafficking in end-membrane system by screening for mutant phenotype

Question 41

what is yeast studied with

Answer 41

conditional mutants

Question 42

what is evolutionary conserved in mutant phenotype analysis

Answer 42

vesicle trafficking/endomembrane organization

Question 43

secretory sec mutants

Answer 43

collection of temperature sensitive mutants that secrete proteins at permissive temperature not at a higher nonpermissive temperature

Question 44

what do sec yeast mutants accumulate

Answer 44

normally secreted proteins at points in the end-membrane pathway blocked by mutation

Question 45

what do sec yeast mutant possess

Answer 45

defects in organelle morphology

Question 46

last thing that sec yeast mutants do

Answer 46

distribution

Question 47

class A of yeast sec mutants

Answer 47

accumulation of secretory proteins in cytosol

Question 48

class B of yeast sec mutants

Answer 48

accumulation in ER

Question 49

how does class A of sec yeast mutants work

Answer 49

defect in protein co-translaton/translocation

Question 50

what causes the accumulation of ER for class B of sec yeast mutants

Answer 50

defect in ER vesicle formation

Question 51

what sec yeast mutants indicate the order of steps in the pathway

Answer 51

double mutants (any combo of A through E)

Question 52

what indicate events involved in biogenesis

Answer 52

other mutants in organelle morphology