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MCB 2050 > Endomembrane System Part 1 - Intro and Methods of Analysis > Flashcards

Endomembrane System Part 1 - Intro and Methods of Analysis Flashcards

1
Q

what do transport vesicles do

A

exchange large amounts of material between each organelle/structure

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2
Q

what do vesicle coat proteins do

A

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

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3
Q

what occurs in the biosynthetic pathway

A

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

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4
Q

what are the 2 types of secretion

A

constitutive
regulated

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5
Q

what occurs in constitutive secretion

A

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

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6
Q

exocytosis

A

vesicle trafficking to and fusion with pm and release of contents

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7
Q

what are secretory vesicle membrane components incorporated into

A

pm - lumenal cargo proteins released into extracellular space

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8
Q

where does regulated secretion occur

A

only specialized cells

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9
Q

what is stored in secretory granules

A

ER-derived materials from Golgi

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10
Q

what do secretory granules do in response to cellular signal

A

fuse with pm and release lumenal cargo into extracellular space

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11
Q

what do secretory granule membrane components get incorporated into

A

pm

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12
Q

what pathway operates in the opposite direction of secretory pathways

A

endocytic pathway - materials move INTO cell

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13
Q

endocytosis

A

uptake of materials from pm and extracellular space into transport vesicles

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14
Q

in the endocytic pathway, where do materials get transported to

A

endosomes and lysosomes

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15
Q

what did autoradiography and pulse-chase radiolabeling experiments demonstrate

A

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

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16
Q

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

A

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

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17
Q

results of brief chase (3 min)

A

rough ER (site of protein synthesis)

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18
Q

results of intermediate chase (20 min)

A

Golgi (site of protein modification)

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19
Q

results of long chase (120 min)

A

secretory vesicles (including those fused with pm)

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20
Q

how did recombinant gene fusion introduce into selected organism/cell

A

via cloning

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21
Q

how does live-cell imaging using autofluorescent proteins work

A

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

22
Q

live-cell imaging using autofluorescent protein result

A

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

23
Q

what is subcellular fractionation

A

isolation of organelle by centrifugation - size/density

24
Q

what is homogenization

A

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

25
Q

what is homogenate

A

filtered and subjected to differential centrifugation

26
Q

what does homogenate remove

A

unbroken cells and large fragments

27
Q

what does differential centrifugation separate

A

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

28
Q

2 components of differential centrifugation

A

pellet at bottom
supernatant

29
Q

what is supernatant

A

liquid at the top of the centrifuge tube

30
Q

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

A

nuclei is isolated in pellet

31
Q

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

A

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

32
Q

what are microsomes

A

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

33
Q

what can individual organelles in each pellet fraction endure

A

can be further purified

34
Q

what does equilibrium density gradient centrifugation separate

A

intact organelles/cellular components on the basis of density

35
Q

cell-free system

A

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

36
Q

liposomes

A

artificial, spherical vesicles consisting of phospholipid bilayer surrounding aqueous centre

37
Q

what do liposomes get mixed with in cell-free systems

A

purified proteins

38
Q

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

A

study of proteins in vitro in natural membrane lipid environment

39
Q

what do cell-free systems allow for

A

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

40
Q

what does mutant phenotype analysis approach to identify

A

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

41
Q

what is yeast studied with

A

conditional mutants

42
Q

what is evolutionary conserved in mutant phenotype analysis

A

vesicle trafficking/endomembrane organization

43
Q

secretory sec mutants

A

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

44
Q

what do sec yeast mutants accumulate

A

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

45
Q

what do sec yeast mutant possess

A

defects in organelle morphology

46
Q

last thing that sec yeast mutants do

A

distribution

47
Q

class A of yeast sec mutants

A

accumulation of secretory proteins in cytosol

48
Q

class B of yeast sec mutants

A

accumulation in ER

49
Q

how does class A of sec yeast mutants work

A

defect in protein co-translaton/translocation

50
Q

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

A

defect in ER vesicle formation

51
Q

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

A

double mutants (any combo of A through E)

52
Q

what indicate events involved in biogenesis

A

other mutants in organelle morphology

MCB 2050 (11 decks)

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