Part B: Lecture 9 + 10 + 11

Question 1

Ahmed paper summary

Answer 1

-identify two gene recruitment sequences (GRS I and II) from promoter of INO1 gene to target gene to nuclear periphery
-mechanism is highly conserved over evolution

Question 2

Blobel proposed the ______ which stated that _____

Answer 2

-gene gating hypothesis
-actively transcribed genes are tethered to NPC

Question 3

silenced heterochromatin is bound to the ________ but abruptly interrupted at the nuceloplasmic face of the NPC by the ______

Answer 3

-inner nuclear membrane
-presence of transcriptionally active euchromatin

Question 4

Nups aka ____

Answer 4

nucleoporin

Question 5

Nups aka ____

Answer 5

nucleoporin

Question 6

yeast is a good model organism because _____

Answer 6

you can change growth media/environment/nutrient source to turn on/off genes

Question 7

Turned on genes move to ____

Answer 7

NPC

Question 8

Localization of a gene in nucleus can be examined in a living cell using a ____

Answer 8

tattooing procedure

Question 9

chromosome tattooing summary

Answer 9

-GFP-tag on protein that binds to gene (ex. Lac repressor -> binds to Lac O array repeats)
-real-time fluorescence

Question 10

Nup49-GFP illuminates the _____

Answer 10

nuclear periphery

Question 11

INO1 gene function? turned on/off when?

Answer 11

-generation of inositol, important in. lipid genesis
-turned on in inositol-depleted media
-turned off in inositol-rich media

Question 12

GRS I in promoter of INO1 gene is ____

Answer 12

8 bp long and targets to NPC

Question 13

Interestingly, introduction of the grs I mutation into the promoter of the endogenous INO1 gene did not ______. This means that there is a ______

Answer 13

• block targeting of the endogenous INO1 to the nuclear periphery
• second targeting sequence upstream of the INO1 promoter, termed GRS II (not related to GRS I)

Question 14

upstream of GRS I is the ____

Answer 14

GRS II

Question 15

mutant GRS I & II = _____

Answer 15

no association with nuclear periphery

Question 16

TSA2 gene encodes an ______ that is activated by ________

Answer 16

-inducible thioredoxin peroxidase
-heat shock and oxidative stress

Question 17

_______ were dispensable for peripheral targeting of INO1. However, most of the proteins associated with the ______ were required for peripheral localization of INO1.

Answer 17

-Most proteins that make up the core channel of the NPC
-nucleoplasmic face of the NPC

Question 18

GRS I from INO1 and TSA2 is related to a sequence motif that was previously found to be _____

Answer 18

overrepresented in promoters of NPC-associated genes

Question 19

INO1 mRNA levels were quantified by _______ following induction by _______ from strains having either plasmid-borne _____ integrated at URA3.

Answer 19

-reverse transcription-qPCR
-inositol starvation
-wildtype (WT) INO1 or grs I mutant INO1

Question 20

GFP-Sec61 is a marker for ______

Answer 20

ER/NE

Question 21

changes to nuclear envelope during metaphase

Answer 21

post-translational modification drive disassembly done by phosphorylation

Question 22

changes to nuclear envelope during anaphase/telophase

Answer 22

chromatin interactions with the nuclear envelope are re-established

Question 23

phosphorylation of lamins and NPC proteins does what?

Answer 23

-disrupts a lot of protein-protein interactions
-forms nuclear envelope fragments during prophase

Question 24

dephosphorylation of lamins and NPC proteins does what?

Answer 24

-promote assembly of nuclear fragments into nucleus
-nuclear envelope fusion during telophase

Question 25

open mitosis def. + type of cells

Answer 25

-NE disrupted -eukaryotic cells + metazoan cells

Question 26

closed mitosis def. + type of cells

Answer 26

-NE stays intact -yeast cells

Question 27

_______ can be used to examine changes in nuclear envelope and chromatin interactions during mitosis because ______

Answer 27

-S. cerevisiae -it uses closed mitosis; NE stays intact

Question 28

NE is continuous with _____

Answer 28

ER

Question 29

GFP Tel 14 is a marker of ____

Answer 29

NE

Question 30

GFP INO1 is a marker of ____

Answer 30

NPC

Question 31

_______ is a positive regulator of post mitotic nuclear envelope reassembly

Answer 31

SUMOylation

Question 32

SUMO aka _____

Answer 32

Small ubiquitin-like modification (15kDa); similar to ubiquitin but doesn't cause degradation

Question 33

SUMO cycle

Answer 33

-SUMO E1 activating enzyme binds to SUMO then transfers to SUMO E2 -SUMO E2 conjugating enzyme; donor of SUMO to other targets -SUMO E3 is a ligase that links SUMO from SUMO E2 to target -target-SUMO has SUMO removed by SUMO proteases

Question 34

parts of SUMO E1

Answer 34

Uba2, Aos1

Question 35

parts of SUMO E2

Answer 35

Ubc9

Question 36

SUMO E3 ligase names

Answer 36

-Siz1, Siz2, Mms21

Question 37

SUMO proteases/isopeptidases names

Answer 37

Ulp1, Ulp2

Question 38

SUMOylaFon at the nuclear envelope ______ during mitosis.

Answer 38

increases

Question 39

The increase in SUMOylation at the nuclear envelope coincides with _______ to the nuclear envelope during mitosis.

Answer 39

Siz2 recruitment

Question 40

The mitotic enrichment of SUMOylation at the nuclear envelope is dependent on _____._____ is enriched at the nuclear envelope during mitosis. The mitosis specific SUMOylation of several proteins is dependent on ___.

Answer 40

-Siz2

Question 41

Clb2 is a tracker of ____

Answer 41

mitosis

Question 42

Loss of Siz2 = ____

Answer 42

loss of SUMOlyation

Question 43

Phosphorylation of Siz2 at _______ is required for recruitment to the nuclear envelope during mitosis.

Answer 43

serine residue 522

Question 44

Scs2 is a _______

Answer 44

-a VAP (VAMP-associated protein) family member -tail-anchored integral membrane protein present in the ER (SUMO site in cytoplasm) and the inner nuclear membrane (SUMO site in nucleoplasm)

Question 45

Scs2 is used as a ____

Answer 45

hook for other proteins

Question 46

General functions of Scs2

Answer 46

-ERAD, deubiquination -lipid droplet -plasma membrane tether of actin -mitochondria tether

Question 47

Scs2 SUMO site is where?

Answer 47

-before the transmembrane region (C-terminal) -SUMO site: VKKE (Val-Lys-Lys-Glu); SUMO is added here

Question 48

Scs2 N-terminus

Answer 48

MSP = + a.a residues; interacts with FFAT motif

Question 49

_____ is nuclear envelope receptor for Siz2 during mitosis.

Answer 49

Scs2

Question 50

Scs2, Siz2 interaction in late mitosis

Answer 50

-Siz2 is phosphorylated & SUMOlyates Scs2 -Siz2 FFAT motif interacts with Scs2 MSP motif (+++) -SUMO site of Siz2 adds SUMO to Scs2

Question 51

end of mitosis: Scs2 remove ____; Siz2 _____

Answer 51

-SUMO -dephosphorylated

Question 52

Recruitment of Siz2 to the nuclear envelope coincides with the reestablishment of _____

Answer 52

chromatin interactions with the nuclear envelope

Question 53

Siz2-mediated SUMOylation at the nuclear envelope facilitates the reassociation of ______

Answer 53

telomeres to the nuclear envelope during mitosis

Question 54

____ is required for the association of telomeres with the nuclear envelope during mitosis

Answer 54

-Sir4

Question 55

Nuclear envelope recruitment of ____ and SUMOylation of ____ are required for telomere association with the nuclear envelope.

Answer 55

-Siz2 -Sir4

Question 56

chromatin IP (CHIP)

Answer 56

-pull down chromatin to see proteins associated with DNA

Question 57

In late mitosis, a cascade of _____ faciliates the re-assoaction of chromatin to nuclear envelope during mitosis + association of proteins associated with NE

Answer 57

SUMOlyation