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MT1 stuff Flashcards

1
Q

Fab region of antibody

A

binds to specific antigens

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2
Q

Fc region of antibody

A

(constant) binds to protein A from bacteria

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3
Q

dounce homogenizer function

A

lyse cells but protect organelle membranes

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4
Q

immunoprecipitation

A

isolate single proteins

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5
Q

Subcellular fractionation: 600xg centrifuge

A

pellet = nucleus
supernatant = everything else

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6
Q

Subcellular fractionation: 15,000xg centrifuge

A

pellet = lysosomes, mitochondria, ribosomes
supernatant = ER, Golgi, PM

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7
Q

Subcellular fractionation: 100,000xg centrifuge

A

pellet = ER, Golgi, PM
supernatant = cytosol

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8
Q

Translocation steps (start to ER docking)

A

Signal protein from ribosome to go to ER lumen
1. free SRP(GDP) in cytoplasm w/ low affinity for SR searches for ribosome w/ hydrophobic signals

  1. when the SRP(GDP) finds the signal sequence exiting ribosome, the ribosome acts as a GEF and makes SRP(GDP) into SRP(GTP). now it has high affinity for SR and translation is arrested.
  2. SRP(GTP) can be a GEF for SR allowing them to bind at ER membrane.
  3. Once bound together, SRP/SR (in GTP states)/ribosome know that they are at ER membrane.
  4. The final step is to transfer ribosome to translocon (pore) and release SRP (via GAP from translocon hydrolyzing GTP–>GDP), once SRP released the ribosome can resume translation
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9
Q

N-linked Glycosylation (when does this happen and what happens)

A

glycosylation is the addition of sugars on proteins on their way to the ER lumen before they fold

  1. on ER membrane, the precursors are assembled (2 NAG’s and 5 mannoses) and span the membrane
    - tunicamycin inhibits precursor assembly
  2. the spanned precursor flips across membrane
  3. 4 more mannoses and 3 glucoses are added
  4. all sugars are added to peptide right after it enters lumen.
  5. after the sugars are transferred to asparagine, three glucoses are chopped
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10
Q

Vesicle transport (budding & docking)

A

Big picture steps
1. ER membrane starts to bud and coat proteins cage the face of bud
2. Once fully separated, the vesicle is entirely coated in proteins and move toward Golgi (anterograde)

Microsteps
1. Sar1 GDP in cytosol gets exchanged to GTP (when nearby membrane) by Sec12GEF to expose alpha helix membrane

  1. Sar1GTP inserted into membrane and recruits Sec23&24 for inner coat/Sec13&31 for outer coat. They cage the vesicle as it pinches off ER membrane
  2. Sec23 is a GAP timer for Sar1, hydrolyzes from
    GTP–>GDP and Sar1 falls off before it can dock and fuse to target membrane
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11
Q

Sec12

A

GEF for Sar1 to go from GDP –> GTP
-permanent resident of ER membrane

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12
Q

Sar1

A

recruits coat proteins for budding in GTP state

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13
Q

Vesicle transport (fusion) & possible problems w/ time

A

v-SNARES find correct T-SNARES to tether and twist to fuse vesicle to target membrane

-too much time: non-specific docking
-too little time: fails to dock

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14
Q

Rab proteins

A

mediate membrane specificity and docking

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15
Q

BiP

A

chaperone that resides in ER lumen and travels in COPII vesicle
-has N-term signal sequence to target translocation to ER lumen.
-has another signal to target for Golgi

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16
Q

KDEL

A

ER return signal at C-term of Bip
-has a receptor that packages proteins into COPI vesicles

17
Q

vesicle transport model (golgi)

A

proteins move in VESICLES to the cisternae

18
Q

cisternae maturation model (golgi)

A

vesicles bring golgi enzymes BACKWARDS to cis golgi and congregate to make new cisternae, old cisternae break apart at end to be recycled

19
Q

LDL

A

low-density lipoprotein, carrier of cholesterol

20
Q

What protein is the address to lysosome?

A

mannose-6-phosphatase, caused by NAG phosphotransferase?

21
Q

GGA

A

mediates interaction of M-6-P receptor and clathrin

22
Q

Arf1

A

like Sar1, clathrin-coated vesicle formation

23
Q

NPXY

A

brings in LDL

24
Q

what is nuclear localization signal

A

PKKKRKV and it’s necessary for function

25
Q

Ran

A

G-protein for nuclear/cytoplasmic trafficking
- RanGTP = in nucleus
- RanGDP = in cytoplasm

26
Q

RCC1

27
Q

nuclear import steps

A
  1. PKKKRKV binds to both importins and can pass through NPC
  2. Once in nucleus, RanGTP disassembles importin complex and cargo is free
  3. RanGTP binds to importins and leaves nucleus to be recycled
  4. Once in cytoplasm, RanGAP activates Ran to hydrolyze GTP –> GDP, RanGDP can’t bind to importins and cycle repeats
28
Q

UNC-83

A

specific KASH protein

29
Q

UNC-84

A

specific SUN protein

30
Q

SUN proteins

A

(type 2 membrane) inner nuclear membrane that is half of bridge across nuclear envelope

31
Q

KASH proteins

A

C-tail anchored proteins that are inserted into ER membrane after translation

BIS104 (2 decks)

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