BIO230 > Lecture 6 > Flashcards

Lecture 6 Flashcards

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1
Q

define glycosylation

A

adding sugar chains to proteins

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2
Q

give two examples of proteins that are glycosylated

A

most soluble and transmembrane proteins in the ER

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3
Q

describe two types of glycosylation

A
  1. O-linked glycosylation (10% of cases): adding sugar chain onto the oxygen of an AA side chain
  2. N-linked glycosylation (90% of cases): adding sugar chain to nitrogen of an asparagine side chain
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4
Q

how is an N-linked oligosaccharide formed?

A
  1. in the ER lumen, an N-linked oligosaccharide precursor is transferred by an oligosaccharyl transferase to an Asn on a protein being synthesised
  2. 3 glucoses are removed (linked to proper folding of the protein), 1 mannose is removed, and a glycosylated protein is transported via vesicles to the Golgi
  3. processing in the Golgi occurs depending on which cisterna you go to.
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5
Q

which side are proteins glycosylated on?

A

only the ER lumen side

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6
Q

describe the sequence rule for where N-linked glycosylation can occur on a protein

A

Asn-X-Ser or Asn-X-Thr where X is any amino acid except proline

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7
Q

describe the structure and resulting functions of the Golgi

A
  • Cis, Medial, and Trans cisternae in Golgi each have different enzymes
  • remove or add sugars, resulting in different modifications to different proteins
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8
Q

describe the removal of glucose and mannose from N-linked oligosaccharide precursors

A
  1. glucosidase I removes one glucose molecule from the precursor
  2. glucosidase II removes another glucose molecule from the precursor
  3. calnexin (a chaperone) binds to the remaining glucose and helps the protein fold
  4. glucosidase II removes another glucose
  5. if the protein is properly folded, ER mannosidase will remove a mannose molecule and the N-linked oligosaccharide will exit from the ER
  6. if the protein is incompletely folded, glucose transferase will add another glucose back onto the chain and the cycle will be repeated
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9
Q

4 functions of glycosylation

A
  • tag to mark the state of protein folding
  • protect proteins on the cell surface from proteases
  • some glycosylated proteins have a role in cell adhesion
  • allows proteins to form the correct 3D structure
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10
Q

3 steps of vesicle movement between compartments

A
  1. budding with cargo
  2. fusion to target
  3. release cargo
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11
Q

two examples of cargo proteins

A
  • transmembrane proteins
  • soluble proteins
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12
Q

how are some soluble proteins bound to vesicles?

A

by transmembrane cargo receptors, which are Y shaped to grab onto the cargo

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13
Q

what is the role of protein coats in vesicle budding?

A

nascent transport vesicles have protein coats:
- select cargo for vesicle
- give curvature to vesicle
- promote vesicle budding

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14
Q

COPI-Coated vesicles

A
  • from Golgi to ER
  • between different Golgi cisternae
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15
Q

COPII-Coated vesicles

A
  • from ER to Golgi
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16
Q

Clathrin-Coated Vesicles

A
  • from Golgi apparatus and plasma membrane to endosome
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17
Q

monomeric GTPases cycle between

A

GDP-bound (off)
GTP-bound (on)

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18
Q

monomeric GTPases are regulated by

A

GEF (guanine nucleotide exchange factor) - turns GTPase on
GAP (GTPase-activating protein) - turns GTPase off

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19
Q

describe the general steps in coat assembly and vesicle formation

A
  1. GEF at site of membrane budding recruits GTPase (GTP-bound)
  2. GTP-GTPase recruits coat proteins
  3. vesicle bud formation, cargo selected
  4. vesicle buds off
  5. vesicle uncoating
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20
Q

how do COPII-coated vesicles form?

A
  1. Sar1-GEF in the ER membrane recruits Sar1-GDP (‘release GDP, get GTP’)
  2. when GTP bins to Sar1, forming Sar1-GTP, an amphipathic alpha-helix is exposed and interacts with the membrane
  3. once anchored, Sar1-GTP recruits coat protein subunits
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21
Q

how do COPI and clathrin-coated vesicles form?

A
  1. ARF-GEF in the Golgi membrane recruits ARF-GDP (‘release GDP, get GTP’)
  2. when GTP bins to ARF, forming ARF-GTP, an amphipathic alpha-helix is exposed and interacts with the membrane
  3. once anchored, ARF-GTP recruits coat protein subunits
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22
Q

describe the two layers of vesicle coats

A
  • inner layer: binds to membrane and selects cargo
  • outer layer: associates with the inner layer to promote polymerisation of the coat (sometimes also selects cargo)
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23
Q

three things that coat proteins need to select

A
  • cargo (transmembrane proteins)
  • transmembrane cargo receptors (bind soluble cargo proteins)
  • SNAREs
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24
Q

describe the structure of COPI-coated vesicles

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A

inner: 4 subunits
outer: 3 subunits

25
describe the uncoating of COPI-coated vesicles
1. γ-COP bins to Arf GAP 2. GTP hydrolysis occurs, causing Arf-GTP -> Arf-GDP 3. Arf-GDP detaches from membrane and coat is released
26
describe the structure of COPII-coated vesicles
inner: 2 subunits (Sec23/Sec24) outer: 2 subunits (Sec13/Sec31)
27
describe the uncoating of COPII-coated vesicles
1. Sec23 - which has GAP activity - is stimulated by Sec13/31 2. GTP hydrolysis occurs, causing Sar1-GTP -> Sar1-GDP 3. Sar1-GDP detaches from membrane and coat is released
28
describe the structure of clathrin-coated vesicles
inner: different adaptor complexes outer: clathrin (6 subunits)
29
what do clathrin-coated vesicles require in order to pinch off?
pinching off of vesicle requires dynamin (has GTPase activity)
30
what do clathrin-coated vesicles require in order to perform uncoating?
uncoating requires Hsp70 and auxillin
31
describe a clathrin molecule
triskelions polymerise to form a curved lattice - 3 heavy chains, 3 light chains
32
what is the specificity of vesicles to target membranes determined by?
1. proteins for docking and tethering the vesicle to the target membrane: Rab GTPases (monomeric) and Rab effectors 2. proteins for catalysing vesicle fusion with the target membrane: SNAREs (transmembrane proteins)
33
how do Rab GTPases and Rab effectors mediate vesicle docking?
1. Rab-GTP binds Rab effector (large variety of Rab effectors) 2. Rab-GTP and Rab effector dock and tether the vesicle. This positions the vesicle close enough for the v-SNAREs (on vesicle) and t-SNAREs (on target membrane) to align. 3. v-SNARE and t-SNAREs bring membranes close together, displace water, and promote membrane fusion
34
binding of t-SNAREs and v-SNAREs promotes
membrane fusion
35
is the interaction between v-SNAREs and t-SNARES specific?
yes; there are many different types of SNARE, but there is specificity in the interaction
36
describe the structural chemistry underlying the v and t SNARE interaction
helical domains coil around each other, locking the two membranes together
37
how can we dissociate the SNARE complex?
NSF and adapter proteins unravel helical domains of vesicles
38
structure of the Golgi apparatus
ER cis Golgi network (CGN) cis cisterna medial cisterna trans cisterna trans Golgi network (TGN) rest of cell
39
how is the budding of vesicles from ER exit sites ensured?
- cargo often has exit signals, leading to the binding of soluble proteins by cargo receptors and the binding of transmembrane proteins by the COPII coat - other cargo has no exit signal, but are packaged because of high concentration in the ER
40
how does cargo go from the ER to the cis Golgi network?
1. COPII-coated vesicles bud from the ER exit site, then shed their COPII coat 2. they fuse with each other to form vesicular tubular clusters 3. vesicular tubular clusters move to the Golgi apparatus
41
define retrieval (retrograde) transport
when vesicles from vesicular tubular clusters and the Golgi go to the ER
42
what proteins is retrieval transport used for?
- escaped ER resident proteins - proteins involved in vesicle budding from ER
43
what does retrieval (retrograde) transport use?
COPI-coated vesicles
44
how do vesicles know which proteins to retrieve?
many ER resident proteins have ER retrieval signals
45
retrieval signal of soluble ER proteins
- soluble ER proteins have a retrieval signal (KDEL) and are bound by the KDEL receptor - they are then packaged into COPI-coated transport vesicles
46
retrieval signal of ER membrane proteins
- eg KKXX at C-terminus - the signal is then bound by COPI coats and packaged into vesicles
47
describe the cycling of KDEL receptors between the ER and Golgi
- at the Golgi, KDEL receptors have high affinity for KDEL (lower pH) - at the ER, KDEL receptors have low affinity for KDEL (higher pH) - this is regulated by pH (V-type ATPase H+ pump)
48
if not all ER and Golgi resident proteins have retrieval signals, how do proteins end up in the right compartment?
1. different transport rates (eg some Golgi enzymes cycle between the ER and Golgi, but transport to the ER at a slower rate) 2. proteins retained in resident compartment (proteins that function in the same compartment form large complexes, which prevents packaging into transport vesicles)
49
how are transport vesicles kept close to the Golgi cisternae?
by tethering proteins
50
name two models for the transport of proteins through the Golgi cisternae
Vesicular transport model cisternae maturation model
51
vesicular transport model
- COPI-coated transport vesicles with argo move forward from one Golgi cistern to the next - retrograde transport vesicles (COPI) return escaped resident ER proteins and Golgi enzymes
52
cisternal maturation model
- cisternae move through the Golgi apparatus - vesicular tubular clusters from the ER fuse to become the cis Golgi network - each cisterna becomes the next cisterna - existing trans cisterna moves to the TGN - retrograde transport by COPI vesicles moves Golgi enzymes and ER resident proteins back
53
are the vesicular transport and cisternal maturation model mutually exclusive?
no, both models may occur: - some cargo move rapidly by transport vesicles - other cargo more more slowly through cisternal maturation
54
define the TGN
a complex network of membranes and vesicles that serves as a major branch point for proteins to be sorted into different vesicles
55
describe the vesicular transport from TGN to the lysosome
- vesicles from TGN carrying lysosomal hydrolases are transported to the late endosomes - late endosomes gradually mature into lysosomes
56
why are lysosomal hydrolases important?
they are needed for lysosome function and degradation of macromolecules
57
acid hydrolyses are synthesised in the --- and processed in the ---
ER; Golgi
58
acid hydrolases are active at ---- pH
acidic
59
1. in the ER, lysosomal hydrolases receive N-linked oligosaccharides. 2. in the cis-Golgi, mannose residues are phosphorylated to form mannose-6-phosphate (M6P). 3. in the trans-Golgi network (TGN), M6P-tagged enzymes bind M6P receptors and are packaged into clathrin-coated vesicles. 4. in the endosome (acidic pH) enzymes dissociate from M6P receptors, which are then recycled to TGN via retromer coat - after dissociation, a phosphatase removes the phosphate group from M6P, which prevents rebinding to the M6P receptor and ensures the enzyme remains in the lysosome. 5. lysosomal hydrolases are activated in the late endosome/lysosome
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