Secretory pathway III

Question 1

What does Golgi resemble

Answer 1

Stack pancakes

Question 2

Is Golgi polar

Answer 2

Yurrrrr

Question 3

Entry face

Answer 3

Cis Golgi face

Question 4

Exit face

Answer 4

Trans Golgi face (sometimes called trans Golgi network)

Question 5

Inside Golgi

Answer 5

Cargoes can be modified by enzymes
Part of reason for Golgi structure = compartmentalizations these functions

Question 6

Name all parts of Golgi in order

Answer 6

Cis Golgi network - sorting, phosphorylation of oligosacc on lysosomal proteins
Golgi stack = cis cisternae (removal of man), medial cisternae (removal of man, addition of glcnac), trans cisternae (addition of gal, addition of nana sialic acid)
Trans Golgi network = sulfation of tyrosines and carbohydrates
(Trans cisternae, trans Golgi network = clip inactive proteins, make active, important for lysosomal enzymes like digestive enzymes)

Question 7

Name all functions of golgi

Answer 7

Sequential modification of n linked oligosaccharides
O linked glycosylation
Proteoglxan glycosylation
Proteolytic modifitications of proteins - late Golgi - tgn
Sorting of proteins towards various destinations = late Golgi tgn
Production of secretory granules - late Golgi

Question 8

Define glycosylation

Answer 8

Reaction in which a carb or chain of chains (glycosyl donor) is attached to a hydroxyl or other functional group of another molecule (glycosyl acceptor)

Question 9

In biology glycosylation =

Answer 9

Refers to enzymatic process that attaches glycans to proteins, lipids or other molecules
- often glycoslayte lipids too

Question 10

How are glycosylation Types classified

Answer 10

According to identify of atom of the a which binds carbohydrates chain - c linked, n linked or o linked

Question 11

Describe c glycosylation And n glycosylation

Answer 11

C glycosylation (not that common) and initial n glycosyaltion takes place in er
Modifications of n glycosylations happen in er and Golgi

Question 12

Describe o glycosylation

Answer 12

Takes place entirely in Golgi

Question 13

Describe glycosylation = all steps

Answer 13

Initial high mannose oligosaccharides into mature complex oligosaccharides
Mannoses and glucoses = involved in quality control
Trimmer —> adds other sugars = glcnac —> then Linked oligosaccharid becomes resistant to removal by endo h = sign protein has gone through Golgi
At end = add Sialic acid = becomes charged, in trans Golgi
(N linked = on asparagine)
If cannot glycoslayte proteins = die
But these steps not essential in tissue culture

Question 14

Describe Golgi enzymes that Modify n linked oliosaccharides

Answer 14

Found in Golgi in same exact sequence as they should act

Question 15

Describe what enzyme stains show

Answer 15

Various Golgi enzymes are distributed only in part in Golgi - specific for each enzyme

Question 16

What is visual sign of trans Golgi

Answer 16

Looks like cisternae pulling off = sign its trans golgi

Question 17

Where does o linked glycosylation Occur

Answer 17

In medial Golgi apparatus
(Most proteins that stay in er are not n linked glycoslayted, happens to almost all proteins that leave er)

Question 18

Describe o liked glycosylation

Answer 18

N acetyl galactosamine linked to serine - often just before glycine or to threonine
- cell type dependent
Other sugars then added
In proteoglycans = process can contribute until chain of 100+ repeating saccharides unit created
Chains can be further modified in Golgi by sulphonation

Question 19

What do secreted proteogylcans do

Answer 19

Form large portion of ecm

Question 20

Describe ph of trans Golgi network

Answer 20

Slightly acidic - ph 6.0 approx, varies by cell type

Question 21

What is found in tgn

Answer 21

Some acid proteases like furin
These proteases process some secreted or lysosomal proteins into Mature/active forms
Dangerous if piece clipped off = happens here
Proteolytic cleave = last step in activating enzymes, right before leave Golgi

Question 22

What does acidic ph in tgn also drives

Answer 22

Aggregation of some secreted proteins - like pancreatic digestive enzymes
Incorporated into secretory granules at high density
Further drop in ph in secretory granules (such as pancreatic condensing vacuoles) can lead to further aggregation, and incorporation of the proteins to be secreted into minimal possible volume

Question 23

Describe organization Golgi = visually

Answer 23

Cisternae connect when Golgi has high rate secretion
Long ribbons with many stacks - if use super res imagining - can see cis medial and trans Golgi
Connecting stacks maybe

Question 24

Describe Golgi structure - stacks

Answer 24

Polar - cis enters and trans exit face
Stacks consisting of layered cisternae
Stacks arranged in long ribbons with disorganized areas between stacks

Question 25

Describe golgi enzymes

Answer 25

Most notably = ones that modify n linked oligosaccharides afre localized sharply to particular regions of Golgi apparatus Enzymes = in right order,but some overlapping distributions

Question 26

Describe vtc - what does cargo avoid

Answer 26

Cargo proteins avoid cop1 pits on vtcs But cargo repcetors are concentrated in cop1 pits Might have role of moving Golgi enzymes in Golgi Golgi and vtc = cop1

Question 27

Describe cop1 on Vtcs

Answer 27

Found in vtcs and Golgi rims -especially cis Golgi Responsible for Golgi/vtc to er retrograde transport, of cargo receptorss

Question 28

What is cop1 composed of

Answer 28

7 polypeptide chains = Alpha, beta, beta prime, gamma, delta, epsilon, zeta Assemble after synthesis into a single permanent stricture (Unlike layered subunits found in cop2 and clathrin coats)

Question 29

Describe cop1 subunits = beta, gamma, delta, zeta

Answer 29

Contain homologies to ap1 (inner subunit cop2) Subunits bind small gtpase arf1 and may correspond to inner subunits of other coats

Question 30

Describe cop1 subunits = beta prime,alpha, epsilon

Answer 30

Have cage forming properties and probably drive membrane curvature Corresponding functionally to outer subunit cop2 or to clahtrin Buttttttt = outer cage that appears to recognize kk Motifs on cargo (cop1 binding signal)

Question 31

Describe arf1 and cop1 steps = step 1

Answer 31

Arf1 gdp in cytoplasm loaded to membrane by gef (gtp exchange factor), also exchanges gdp for gtp Recruited to membrane,has small fatty acid chain that is inserted

Question 32

Describe arf1 and cop1 steps = step 2

Answer 32

Arf1-gtp on membrane recruit cop1 which can recruit cargo

Question 33

Describe arf1 and cop1 steps = step 3

Answer 33

COP1 forces membrane curvature and budding

Question 34

Describe arf1 and cop1 steps = step 4

Answer 34

Arfgap1 or arfgap2 = can bind to cop1 vesicles causing arf1 gtp to hydrolzye gtp Arf1 gdp not stable on membrane = os leaves

Question 35

Describe arf1 and cop1 steps = step 5

Answer 35

Cop 1 coat release Leaves membrane = uncoats vesicle

Question 36

Describe arf1 and cop1 steps = describe exchange factors and gap proteins

Answer 36

Exchange factor and gap proteins can be diff for clathrin coated vesicles - also assembled by arf1 Arf1 = regulates recruitment of cop1 coat to Golgi and clathrin coat to other memrbanes

Question 37

Describe questions about cargo receptors

Answer 37

How does cargo know when to bind/release cargo repcetor How is cargo receptors recycled back to er

Question 38

What are signals that Cargo repcetros recognize

Answer 38

Dilysine residue kk But also variations like qk

Question 39

Describe what most cargo receptors do

Answer 39

Bind carg in er and release in Golgi/vtc = most besides kdel

Question 40

Describe what kdel does

Answer 40

Binds escaped proteins in Golgi and returns it er = proteins that are not supposed to leave er, resident proteins which leave er at slow rate, most resident proteins have kdel = binds repcetros

Question 41

Describe sequences on most cargo repcetros

Answer 41

Sequence that interacts with cop2 - diphenylalanine ff at c term = allows exit from er Have sequence that interacts with cop1 - often kk or variation (glutamine, Lysine) = allows return from vtc or Golgi to er

Question 42

Describe interaction with cargo of most cargo repcetros - ph

Answer 42

Have ph sensitive interaction with cargo = bind cargo at neutral ph - er lumen 7.5and lose binding at slightly acidic ph - 6.5 in Golgi or vtc (Also calcium concentrations in er important)

Question 43

Describe Ergic53

Answer 43

Some proteins with Tm domains can interact with it Binds soluble cargo proteins with high mannose oligosaccarides - signal to leave er Vip35 and vipl = 2 related proteins that may have similar function Binds tightly at neutral ph and high ca2+ and lose binding @ low ph and low ca2+

Question 44

Describ p24

Answer 44

Family = binds gp1 anchored proteins

Question 45

Describe surf4

Answer 45

Binds a 3 aa sequence at the n terminus of some soluble cargo proteins

Question 46

Describe exp = ergic53

Answer 46

Binds mannose on n linked oligosaccahrides in er - neutral ph Vtcs - slightly acidic = enables cargo to detach from ergic53

Question 47

Describe specifically how p24 family proteins act

Answer 47

Cargo receptors for gpi anchored proteins Eveidcne shows that p24 binds gpi anchored proteins at neutral ph in er and releases them at slightly acidic ph

Question 48

Where are kdel sequences found

Answer 48

Many resident lumenal er proteins

Question 49

Describe proteins with kdel sequences

Answer 49

Not efficiently recruited to cop 2 vesicles But not excluded = can be caught there on accident at slow rate = resident er proteins can gradually leave er bu accident

Question 50

What is kdel sequence bound by

Answer 50

4 aas = bound by kdel receptor in Golgi apparatus Kdel receptor = then incorporated into cop1 vesicles and returned to er

Question 51

When does kdel receptor bind/release kdel

Answer 51

Binds kdel at acidic ph (vtcs and Golgi = Mildly acidic) Releases kdel at neutral ph (er) Reverse specificity of forward cargo receptors

Question 52

Name things that can Golgi/vtc to er retrograde traffick

Answer 52

Er cagro repcetros Kdel receptors Snares - fusion Enzymes???

Question 53

Describe how enzymes retrograde tarffick

Answer 53

Golgi enzymes cycle through er = bc expose them to er quality control If protein at end of life - worn out = not folding right = brought back to er = recognized as misfolded and can be degraded

Question 54

Describe cop1 pits

Answer 54

Cargo proteins avoid cop1 pits Cargo repcetros conentrated in cop1 pits

Question 55

Describe Rothman = how to go through Golgi apparatus exp QUESTIONS

Answer 55

James Rothman produced an in vitro system to study trafficking through the Golgi apparatus (described in the next few slides). He believed that cargo was transported between cisternae by coated vesicles, and wished to analyze this process. Hypothesis = made in vitro cell free system with 2 pops purified golgis = try and reconstitute trafficking from one pop to other

Question 56

DESCRIBE MEMBRANE FRACTIONATION

Answer 56

To purify Golgi = multiple steps and complicated Differential centrifugation Density gradient centrifugation Multiple fractionation steps Developed in palade lab and elsewhere as means of studying organelles biochemically

Question 57

DESCRIBR isolation of lysosomes - gen

Answer 57

Differential centrrifuagtion used in initial quick steps Final purification involves density gradient centrifugation = common pattern, multiple steps Break cells and spin and pellet nuclei - spin again and pellet mito then final step fo density gradient centifugation

Question 58

Describe one strategy to purify golfi

Answer 58

More complicated Elaborate process Rothman wanted to make sure Golgi still functional = so used membrane fractionation

Question 59

Describe process = membrane fractionation

Answer 59

Multi step process = involves multiple centrifugations and possibly other steps Purification of Golgi established in 1960s to 1970s Rothman lab refined technique = with goal of purifying golgis that preserved function, including cagro transport

Question 60

Describe process = membrane fractionation = what are Golgi fractions used for

Answer 60

As one components of an invitro cell free system to reconstitute cargo transport through Golgi - in test tube

Question 61

Needed for cell free system =

Answer 61

Sufficient components to support process of interest = can be completely purified and characterized molecules or organelles or partially intact cells - atp, cytoplasm, gtp, salt Process which investigator wants to study Asssay = to determine whether and how efficiently desired process took place

Question 62

Needed for cell free system = What type of cytosol used

Answer 62

Uncharacterized cytosol used for initial develop of an in vitro system Later on = effort made to replace total cytosol with purified proteins = allows determination of minimal proteins required for process to occur = try to find minimal amount of proteins needed

Question 63

Why are cell free systems used

Answer 63

Study particular aspects of a process without all of the complexities of a fully intact cell Process will be reduced to minimal components Ex= translocation into er reconstituted using only purified microsomes, cytosol and mRNA

Question 64

What does cell free system allow

Answer 64

Testing potential requirements for process of interest - atp, gtp, proteins In many cases = important proteins can be identified purely by using cell free system as an assay

Question 65

Describe intro system for intragolgi trafficking = the 2 populations

Answer 65

Golgi prepared from 2 pops of Chinese hamster ovary cells by density gradient centrifugation ONE POP =expressed vsv G protein but missing transferase TWO POP = normal but expressed no G protein

Question 66

Describe intro system for intragolgi trafficking = mixing

Answer 66

Mix pops and cytosol and atp added, mg, ca and other things

Question 67

Describe intro system for intragolgi trafficking = results

Answer 67

G protein processed, measured by incorporation of radioactive glcnac = interpreted to mean it was transported from one Golgi pop to other = Rothman interpreted it as G protein moving, but= Move G protein to Golgi with enzyme Move enzyme to Golgi with G protein Or golgis fuse at random - but ruled this out

Question 68

Describe detecting glcnac incorporation

Answer 68

Dissolve cells - with detergent to stop reaction Vsvg was immunoprecipiated from reaction mix with an then wash beads and measure radioactivity Glcnac incorporation detected by sds page = specifically to show its real the glcnac attaching to G protein and everything is going well Once incompatible confirmed = immunoprecipiates radioactivity measured using scintillation counter = gave numerical value for incorporation efficiency

Question 69

Primary assay for Intra Golgi transport = 2 pops

Answer 69

Two populations of Golgi are mixed. One (from mutant cells) contains a cargo protein (the Vesicular Stomatitis Virus G protein) but lack GlcNac transferase. The other Golgis (from wild-type cells) possess GlcNac transferase but do not contain VSV-G

Question 70

Primary assay for Intra Golgi transport = radioactive

Answer 70

Radioactive (tritium-labeled) GlcNac is added along with components (cytosol, ATP, GTP) needed to support transport

Question 71

Primary assay for Intra Golgi transport = incorporation

Answer 71

Incorporation of GlcNac into N-linked oligosaccharides of VSV-G only happens if VSV-G and GlcNac transferase are in the same compartment. This was interpreted as intra-Golgi transport

Question 72

Primary assay for Intra Golgi transport = describe image

Answer 72

G protein = by infection, integral membrane protein on surface of virus —> goes through secretory pathway Has cop 2 binding sequence Results = assumed G protein moved - that had glcnac incorporated into it but actually glcnac move Note = cells lack enzyme that adds glcnac = not present in these cells os complex oligosaccride are abnormal in these cells

Question 73

Describewhat happened after conditions worked our where glcnac incorporation occured

Answer 73

= now cell free assay existed for intra Golgi trafficking Next goral = use assay to determine which proteins in cytosol essential

Question 74

Em examination of in vitro system

Answer 74

Visualization of successful transfer reactions was possible by electron microscopy In these reaction mixtures, coated pits were visible on Golgi. Both coated and non-coated vesicles were identified The coat was unknown. Rothman hypothesized that a sequence of events were required to form a vesicle and fuse it with a target membrane. A goal of the in vitro system was to identify the steps involved

Question 75

Describe further work with cell system = set up exp

Answer 75

Non hydrolyzable analog of gtp - gtp gamma s = added = vesicles coated Reaction wont work Vesicles never uncoat

Question 76

Describe further work with cell system = Purification fo vesicles

Answer 76

Led to identification of several proteins making up coat = COP, b-COP, b’-COP, g-COP, d-COP, e-COP and z-COP. The complex was named “coatomer protein” or COP Stoichiometric = all present in same amount

Question 77

Describe further work with cell system = Proteins discovered

Answer 77

Arf discovered to be gtp requiring component, gdp form in cytoplasm Cop1

Question 78

Describe intra golgi trafficking - 1970s

Answer 78

3 models= 1 - Golgi could be connected in such a way that proteins diffuse through connections between cisternae Or cis cisterna could gradually azure to become the trans cisterna Or each cisternal could be a separate compartments with vesicles required to transport cagro - each has distinct features Idea = cisternal maturation favoured by em = see trans cisternae peeling off But cisternal matruation could not explain how different Golgi enzymes kept segregated in separate cisternae

Question 79

Describe Rothman model = role of coated vesicles in intragolgi trafficking 1989

Answer 79

Through cop1 moves through And enzymes stay in place and do not move

Question 80

Problem = Rothman model

Answer 80

Vesicles contained Golgi enzymes like mannosidase but did not contain cagro Have less G protein than golfi cisternae so didn’t look like G protein using cop1 But = became clear that Rothman was looking at glcna transferase budding off Golgi and delivering enzyme to other Golgi with G protein

Question 81

Describe exp = algal scales

Answer 81

Provide evidence for cisternal matruation. Scales = huge, ended up in big vesicles but this was not representative of mammalian cells

Question 82

Describe direct evidence for cisternal maturation = procollagen

Answer 82

Procollagen only exits er in presence ascorbic acid Add ascorbic acid for short time and let procollagen exit er then remove Procollagen appears first in cis Golgi, medial compartmts then trans Not spread all over Golgi = can see if do immunogold *vit c

Question 83

Describe direct evidence for cisternal maturation = exp 2 in mammalian cells

Answer 83

Ts035 mutant of vsvg misfolds at 40c and is held in er by quality control machinery Can sill fold correctly and leave er if cells shifted to 35c Once out of er - it will continue through secretory patwhay regardless of temp Like procollagen = allowed to leave er for short period of time then go back to 40c = evidence for cisternal maturation Do immunogold= looks like vesicles picking up G protein at random Creates pulse of G protein = saw G protein enter single cisternae at cis golgi and the G protein stayed in that single cisternae as it migrated to trans face = proves cisternae move and cargo stays in same cisternae (If cagro moved = would see G protein spread out and in multiple cisternae)

Question 84

Sooo cisternal maturation…

Answer 84

= true explanation of how most cargos move from cis to trans golg within Golgi apparatus

Question 85

But if cisternae move how are enzymes alwayas in right space

Answer 85

Can only work if enzymes constantly relocating But does not discredit cisternal matruation bc too much evidence

Question 86

Possibility 1 of enzymes in cisternal matruation model

Answer 86

Cop1 vesicles move enzymes backwards in Golgi at Same rate of cisternal maturation

Question 87

Possibility 2 of enzymes in cisternal matruation model

Answer 87

Enzymes move within Golgi - maybe randomly and are localized by other means like selective affinity for their substrates Could work in theory but not shown

Question 88

Is it possibility 1 or 2 - cisternal maturation model

Answer 88

Do not know exactly appear to be different variants of COPI vesicles, but it is difficult to see how they can be fine-tuned to always put the enzymes in the right place If the enzymes can diffuse freely through tubular connections, how are cargo prevented from using the same interconnections? Do they prefer to be in different lipid domains? Resident proteins typically have SHORTER transmembrane domains than cargo integral membrane proteins, so this is not so far-fetched. Also Golgi cisternae glued together by tethering proteins = these could have ability to bind enzymes, have diff lipid domains

Question 89

Does cop1 move proteins between cisternae?? If not what does it definitely do

Answer 89

May or may not = don’t know But have substantial evidence that cop1 is neeeded for recycling of proteins from Golgi to er Proteins that cycle between er and Golgi - cargo receptors are highly concentrated in cop1 pits and vesicles on both Golgi and vtc Proteins have cop1 interacting sequences on cytoplasmic portions Deletion of cop1 subunits in yeast lead to appreance of cargo receptors on surface

Question 90

Cop1 is responsible for

Answer 90

Recycling from Golgi/vtc to er

Question 91

Describe conclusions - intra Golgi trafficking 2010s

Answer 91

Cisternal matruation = now known to be how cargoes transported through Golgi But cisternal matruation still does not explain how diff Golgi enzymes are kept segregated in separate cisternae Both other models - direct connections between cisternae and shuttling of enzymes by cop1 = have some support BUT COMPLICATEDD = could all be right or wrong…

Question 92

What is proteoglycan

Answer 92

Very large secreted proteins that have o linked glycosylation