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Cell 600 > Sept 19 - Golgi Lecture > Flashcards

Sept 19 - Golgi Lecture Flashcards

1
Q

Nocadozole

A

An agent which destabilizes microtubules and causes dispersal of the golgi ribbon. Does not alter golgi function, unlike BFA which disrupts golgi structure and function.

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2
Q

Compact region

A

Contains flattened cisternae

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3
Q

Non-compact region

A

Contains vesicles, tubules

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4
Q

Cis face (CGN)

A

Face closest to the ER

The ER is its “sister”

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5
Q

Trans face (TGN)

A

Secretory vesicles bud off from the trans face

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6
Q

GalT

A

Galactosyl transferase, a trans Golgi enzyme

The T could also stand for trans

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7
Q

EB3

A

End binding protein 3, a microtubule associated protein (the comet one!)

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8
Q

MTOC

A

Microtubule organizing center. The golgi is located near the MTOC.

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9
Q

Mini-Stacks

A

Dispersed golgi membranes

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10
Q

Golgi ribbon

A

Aligned mini-stacks

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11
Q

lmh1

A

TGN protein

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12
Q

Sec7

A

Arf exchanger

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13
Q

Golgins/GRASPs

A

Bind to Rab to tether Golgi cisternae in place

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14
Q

Cis–> Trans

A
  1. Phosphorylation of oligosaccharides
  2. Removal of man
  3. Addition of GlcNAc
  4. Addition of Gal
  5. Addition of NANA
  6. Sulfation of tyrosines and carbohydrates
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15
Q

Cis–> trans for N-linked oligosaccharides

A
  1. Golgi mannosidase I
  2. N-acetylglucosamine transferase I
  3. Golgi mannosidase II
    // ENDO-H SENSITIVE VS RESISTANT
  4. ?
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16
Q

Mannosidase

A

Trim mannose residues

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17
Q

Which golgi enzyme determines Endo H sensitivity?

A

Mannosidase II, which acts in the medial golgi
Glycoproteins in early Golgi are sensitive to cleavage by Endo H, but after they have been modified in the late Golgi, they are resistant

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18
Q

PNGaseF

A

Protein N glycosidase F, an endoglycosidase which cleaves off complete n-linked oligosaccharide regardless of whether or not it is high mannose or complex.

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19
Q

NANAse

A

Neurominidase - a glycosidase that removes neuraminic acid residues, can be used to determine whether a protein has pssed through the TGN

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20
Q

High mannose

A

2 N-acetylglucosamines with many mannose residues

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21
Q

Complex

A

Contains almost any number of other types of saccharides

22
Q

Blocks important in glycolipid biosynthetic machinery?
Honestly, I think both of these also block glycoproteins… I came across some papers saying if you knock these out you get decreased transport all around…

A
  1. Monensin block - blocks transport from medial to trans golgi cisternae
  2. BFA - brefeldin A - blocks COPI formation
23
Q

Brefeldin A

A

The main target of brefeldin A appears to be a Guanine nucleotide exchange factor called GBF1.[4] GBF1 is a Sec7 family of Arf GEFs which prevents further GTP loading of Arf1. It mediates formation of transport vesicles by recruiting COPI coat proteins to cargo-bound receptor proteins found in the membrane of the Golgi. Inhibition of GBF1 activity induced the retrograde movement of secretory proteins from the golgi to the ER.

BFA treatment causes most but not all, GOLGI MEMBRANE PROTEINS TO BE DELIVERED TO THE ER.

DOES NOT AFFECT GOLGI MATRIX - ONLY THE MEMBRANES.

Used in arguments supporting constitutive ER–> Golgi retrograde transport pathway.

24
Q

How does golgi lipid membrane composition change from cis to trans?

A

Complex sphingolipids partition from glycerophospholipids and then are transported to the plasma membrane

25
Q

How can you visualize golgi transport?

A

Express fluorescently tagged, temp sensitive viral glycoprotein from VSV tagged with EGFP.
At 39 degrees, partially unfolded and trapped in ER
At 32 degrees, exits ER in a wave
At 30 minutes, all in the Golgi - colocalizes with TGN46
At 60 min, on cell surface

26
Q

Sar1

A

It is a GTPase found in COPII vesicles. It regulates the assembly and disassembly of COPII coats.

27
Q

Dominant negative Sar1

A

Blocks COPII assembly and therefore ER–> Golgi transport

28
Q

What happens when you

  1. Inject cells with DNSar1
  2. Incubate with cyclohexamide
  3. Stain for Mannosidase II
A

You are basically blocking exit from the ER (because Sar1 blocks COPII assembly and ER–>Golgi transport so you see buildup of golgi enzymes in the ER.
The cyclohexamide makes sure you are focusing only on pre-existing proteins.

29
Q

Cycloheximide

A

inhibitor of protein biosynthesis in eukaryotic organisms, produced by the bacterium Streptomyces griseus. Cycloheximide exerts its effect by interfering with the translocation step in protein synthesis (movement of two tRNA molecules and mRNA in relation to the ribosome) thus blocking translational elongation.

30
Q

What are 2 things contained in COPII vesicles?

A
  1. Anterograde cargo

2. Escaped resident ER proteins (which are later retrieved from the GA by retrograde trafficking).

31
Q

Where do COPII vesicles bud from?

A

ER exit sites

32
Q

Emp24

A

Example of a cargo receptor that binds soluble cargos, allowing it to be placed in COPII vesicle

33
Q

VTCs

A

Vesicular Tubular clusters - Free COPII vesicles fuse to form these. These are transported along microtubules to the golgi.

34
Q

Transitional elements, ERGIC (ER-Golgi Intermediate Compartment)

A

Another word for VTCs

35
Q

Homotypic fusion

A

Fusion of membranes from the same compartment. A symmetric interaction; both contribute their own vsnare and tsnare. This is how VTCs are formed.

36
Q

Are there enough SNAREs to maintain identity for stepwise cis–>trans processing?

A

No - there are only 2 sets of SNAREs, not nearly enough.

37
Q

2 models for Golgi cis to trans maintenance

A
  1. Vesicle transport model / stable compartments: Golgi stacks are static, stable organelles, enzymes localize to a stack and stay put.
  2. Cisternal maturation model: Stacks are not static structures, fusion near MTOC forms a new Cis face of the golgi. Cargo stays put but golgi stack matures by RETROGRADE, vesicle-mediated transport of golgi enzymes.
38
Q

KKXX

A

ER retrieval signal that COPI binds to. 2 lysines + 2 AAs. Found at C-terminus of type I integral membrane proteins that are ER residents (i.e. calnexin)
The fact that COPI binds to KKXX suggests that it is involved in retrograde transport, consistent with the cisternae maturation model.

39
Q

KDel receptor

A

Recognizes ER lumen proteins (BiP, GRP94, protein disulfide isomerase) with C-terminal KDel sequence.
Thought to retrieve ER proteins that accidentally escaped to Golgi.
It has the KKXX sequence on C terminus.

40
Q

Vps74 (yeast) /Gmx33 (mammalian)

A

A COPI adaptor, peripheral membrane golgi protein that binds to COPI and cytoplasmic domain of many Golgi glycosyltransferases but not to ER or vacuole membrane proteins.

In Vps74 mutants, Golgi proteins are mislocalized to the vacuole.

41
Q

BFA versus nocodazole

A

BFA - disrupts golgi structure and function (disrupts Arf GEFs) and inactivates the COPI retrieval system.

Nocodazole - disassembles Golgi ribbon but does not alter its function. Interferes with microtubules.

42
Q

Vrg4

A

Early Golgi GDP-mannose transporter

43
Q

Sec7

A

Late Golgi Arf1 GEF (this is what BFA disrupts)

44
Q

GPI

A

Apical cargo carrier

45
Q

VSVG3

A

Basolateral cargo carrier

46
Q

COG

A

Multisubunit tethering complex required for intra-golgi transport; absence results in distention and fragmentation of Golgi cisternae and mislocalization of Golgi enzymes.

47
Q

CDG patients

A

Multi-system disease with severe neurological involvement.

Congenital disorder of glycosylation.

48
Q

SREBP

A

Sterol responsive element binding protein - transcription factor that regulates genes involved in sterol synthesis and uptake. Want on in low cholesterol conditions.

49
Q

Pro-SREBP

A

how SREBP is synthesized - inactive, membrane bound precursor

50
Q

SCAP

A

The escort protein that proSREBP is bound to in the ER

51
Q

INSIG

A

Protein that retains SCAP-Pro-SREBP complex in the ER through association when cholesterol is abundant. When cholesterol is depleted, INSIG releases SCAP.

52
Q

S1P, S2P

A

Golgi proteases that cleave SREBP and liberate transcription factor moiety

Cell 600 (9 decks)

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