Secretory pathway II

Question 1

Describe Rothman

Answer 1

Biochemical work

Question 2

Describe Sherman

Answer 2

Yeast genetics

Question 3

Name 2 major centrifugation techniques

Answer 3

Differential centrifugation
Density gradient centrifugation

Question 4

Describe differential centrifugation

Answer 4

Achieved by subjecting suspension of cellular components (obtained by disrupting cells by homogenization) = to action of centrifugal forces
= load top of tube with heavy and light things, nuclei go to bottom faster bc heavier, have to freeze tube to stop sinking
1 step = usually get rid of nuclei
Works best with things of diff density

Question 5

Describe differential centrifugation - sedimentation coefficient

Answer 5

Primarily depends on shape and size of particles and organelles

Question 6

Sedimentation coefficient for lysosomes

Answer 6

9400s

Question 7

Sedimentation coefficient for ribosomes

Answer 7

80s

Question 8

Describe density gradient centrifugation

Answer 8

Improvement in technique of differential centrifugation
Gradient made with sucrose or metrizamide
Concentration - density max at bottom and minimal at top
Can be continuous or discontinuous gradients
Make possible purer fractions of organelles
To collect fraction = perforate at bottom

Question 9

Describe density gradient centrifugation - how do things separate

Answer 9

Centrifuge then particles penetrate gradient but only to level where an equilibrium exits between action of centrifugal force and tendency of particle to float
= separation of particles due to their buoyant density = independent of size and shape = penetrate to level of their own density

Question 10

Describe density gradient centrifugation Example

Answer 10

Usually do differential centrifugation first to get ride nuclei and mito etc, need to time this bc will sink further
Then do densiry gradient centrifugation
Homogenization —> tube with gradient of increasing sucrose concentration —> rer with ribosomes winked lowered
Can leave in density gradient centrifugation for long = doesn’t matter bc when get to right desity will stop

Question 11

Describe Schekmans use of density gradient centrifugation

Answer 11

Simpler
- wanted to separated heavy from light yeast
Yeast intact and alive - not in organelles
Not broken open

Question 12

Describe what schekman wanted to do

Answer 12

Determine which proteins important for secretory pathway in yeast

Question 13

Describe exp set up = isolation of temperature sensitive sec mutants = specifically

Answer 13

Yeast exposed to mutagen and grown at 24c = assumed cells would increase in density - more protein if they could not secrete = accumulate
Shifted to 37c to get mutation for 3 hours - extra heavy yeast separated by density gradient centrifugation - separate heavy yeast
Moved back to 24c - if secretion blocked too long = would kill yeast

Question 14

Describe exp set up = isolation of temperature sensitive sec mutants = generally

Answer 14

See what causes secretion to stop
= want to make mutants that will survive tho
So do temp sensitive mutation = get mutants to then characterize colonies
Hard bc need to find mutant we want - must grow yeast in presence mutation and find it but rare

Question 15

Describe exp set up = isolation of temperature sensitive sec mutants = SCREENING

Answer 15

Colonies at 24c screeened and further characterized
Mutations in 23 genes identified in first screen = sec mutants

Question 16

DESCRIBE complementation groups = yeast types

Answer 16

Yeast can be haploid or diploid

Question 17

Describe haploid yeast

Answer 17

Contain only one copy of each gene
No redundancy, no back up if mutation
Can be mated to produce diploid yeast

Question 18

Describe diploid yeast

Answer 18

Have 2 copies of each gene
Can be grown, or under stressful condition undergo meiosis to generate haploid yeast with og genes shuffled
Then can see if diploid yeast have phenotypes

Question 19

Describe exp = haploid wt and haploid mutant dies at 37c

Answer 19

Diploid = survives at 37c bc one of 2 copies in good
(Most mutations loss of function!!!)

Question 20

Describe exp = mutation in same complementation group

Answer 20

SAME GENE
So dies at 37c
Neither copy good, cannot survive
= 2 mutations in same complementation group = code for similar polypeptides in Ismaili dna region, affects same protein
But mutations diff thoooo

Question 21

Describe exp = mutation in diff complementation group

Answer 21

Diff genes
= diploid yeast survives at 37c bc has one good copy of each gene x and gene y as well as one bad copy of each

Question 22

What did sec mutants mostly show - complementation groups

Answer 22

Mostly showed loss of protein activity at non permissive temp = protein coded for by the gene failed to function
= mutations mostly recessive

Question 23

Describe if mutations in same gene- complementation groups

Answer 23

Diploid yeast containing only one copy of mutant gene would be normal
If haploid yeast containing distinct mutations mated = diploid progeny would be normal, even at 37c
But if muttaions in same gene = diploid progeny still temp sensitive

Question 24

How were larger mutations sorted into 23 complementation groups

Answer 24

= seeing which diploid progeny survives

Question 25

Describe further studying the complementation groups

Answer 25

Using ‘em = groups them diff

Question 26

Name the types of mutants - sorted by em

Answer 26

Sorted into groups depending on whether er accumulated at non permissive temps = suggested block in exit from er Golgi membrane acculturated = enlarged yeast golgis first identified - Berkeley bodies Or whether vesicles accumulated = seemed like 3 important parts where trafficking would stop = er, Golgi and Golgi —> surface

Question 27

Describe accumulated er group

Answer 27

Er abnormal amount accumulated Sec12, sec13, sec23 (Also sec24, sec 31 but not identified in first screen) (Also sec16,17,18,20,21,22 but don’t need t know)

Question 28

Describe accumulated golgi in some mutants

Answer 28

Few mutants = sec7, sec 14

Question 29

Describe accumulated vesicles

Answer 29

Assumed that the proteins coded by these genes were required to fuse with pm after leaving Golgi = sec 1,2,3,4,5,6,8,9,10,15

Question 30

Describe discovery cop2 = who

Answer 30

Schekman lab = discovered conditions in which vesicles could be made from purified er

Question 31

Describe discovery cop2 = describe exp

Answer 31

Required incubating er with cytoskeleton, atp, gtp, magnesium, right salt concentrations, other factors

Question 32

Describe discovery cop2 = when are coated vesicles accumulated

Answer 32

When gtp analog introduced that could not be hydrolyzed = vesicles remain coated

Question 33

Describe discovery cop2 = describe technique and exp - immunogold

Answer 33

Cop2 membranes prepared from purified (in vitro) er membranes by blocking gtp hydrolysis = could be tested for presence of sec proteins involved in er to Golgi trafficking by immunogold, identified what is in cop2 coat = sar1, sec23, sec24, sec13, Sec31

Question 34

What did Rothman discover

Answer 34

Coatomer protein 1

Question 35

Describe why schekman lab exp worked

Answer 35

Bc sar1 binds gtp - brought to membrane Exp replaced gtp with gtp mas = guanine, oxygen, phosphate, oxygen, oxygen, phosphates - many o2 And o2 unstable - replace with sulfur So sar1 cannot hydroplane gtp - coat stays on and cannot leave = could work up order of coats

Question 36

Describe relative Densities of some things

Answer 36

Rer denser than ser Lysosomes denser Golgi less dense than er