Part B: Lecture 9 + 10 + 11 Flashcards
Ahmed paper summary
-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
Blobel proposed the ______ which stated that _____
-gene gating hypothesis
-actively transcribed genes are tethered to NPC
silenced heterochromatin is bound to the ________ but abruptly interrupted at the nuceloplasmic face of the NPC by the ______
-inner nuclear membrane
-presence of transcriptionally active euchromatin
Nups aka ____
nucleoporin
Nups aka ____
nucleoporin
yeast is a good model organism because _____
you can change growth media/environment/nutrient source to turn on/off genes
Turned on genes move to ____
NPC
Localization of a gene in nucleus can be examined in a living cell using a ____
tattooing procedure
chromosome tattooing summary
-GFP-tag on protein that binds to gene (ex. Lac repressor -> binds to Lac O array repeats)
-real-time fluorescence
Nup49-GFP illuminates the _____
nuclear periphery
INO1 gene function? turned on/off when?
-generation of inositol, important in. lipid genesis
-turned on in inositol-depleted media
-turned off in inositol-rich media
GRS I in promoter of INO1 gene is ____
8 bp long and targets to NPC
Interestingly, introduction of the grs I mutation into the promoter of the endogenous INO1 gene did not ______. This means that there is a ______
- 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)
upstream of GRS I is the ____
GRS II
mutant GRS I & II = _____
no association with nuclear periphery
TSA2 gene encodes an ______ that is activated by ________
-inducible thioredoxin peroxidase
-heat shock and oxidative stress
_______ were dispensable for peripheral targeting of INO1. However, most of the proteins associated with the ______ were required for peripheral localization of INO1.
-Most proteins that make up the core channel of the NPC
-nucleoplasmic face of the NPC
GRS I from INO1 and TSA2 is related to a sequence motif that was previously found to be _____
overrepresented in promoters of NPC-associated genes
INO1 mRNA levels were quantified by _______ following induction by _______ from strains having either plasmid-borne _____ integrated at URA3.
-reverse transcription-qPCR
-inositol starvation
-wildtype (WT) INO1 or grs I mutant INO1
GFP-Sec61 is a marker for ______
ER/NE
changes to nuclear envelope during metaphase
post-translational modification drive disassembly done by phosphorylation
changes to nuclear envelope during anaphase/telophase
chromatin interactions with the nuclear envelope are re-established
phosphorylation of lamins and NPC proteins does what?
-disrupts a lot of protein-protein interactions
-forms nuclear envelope fragments during prophase
dephosphorylation of lamins and NPC proteins does what?
-promote assembly of nuclear fragments into nucleus
-nuclear envelope fusion during telophase
open mitosis def. + type of cells
-NE disrupted
-eukaryotic cells + metazoan cells
closed mitosis def. + type of cells
-NE stays intact
-yeast cells
_______ can be used to examine changes in nuclear envelope and chromatin interactions during mitosis because ______
-S. cerevisiae
-it uses closed mitosis; NE stays intact
NE is continuous with _____
ER
GFP Tel 14 is a marker of ____
NE
GFP INO1 is a marker of ____
NPC
_______ is a positive regulator of post mitotic nuclear envelope reassembly
SUMOylation
SUMO aka _____
Small ubiquitin-like modification (15kDa); similar to ubiquitin but doesn’t cause degradation
SUMO cycle
-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
parts of SUMO E1
Uba2, Aos1
parts of SUMO E2
Ubc9
SUMO E3 ligase names
-Siz1, Siz2, Mms21
SUMO proteases/isopeptidases names
Ulp1, Ulp2
SUMOylaFon at the nuclear envelope ______ during mitosis.
increases
The increase in SUMOylation at the nuclear envelope coincides with _______ to the nuclear envelope during mitosis.
Siz2 recruitment
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 ___.
-Siz2
Clb2 is a tracker of ____
mitosis
Loss of Siz2 = ____
loss of SUMOlyation
Phosphorylation of Siz2 at _______ is required for recruitment to the nuclear envelope during mitosis.
serine residue 522
Scs2 is a _______
-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)
Scs2 is used as a ____
hook for other proteins
General functions of Scs2
-ERAD, deubiquination
-lipid droplet
-plasma membrane tether of actin
-mitochondria tether
Scs2 SUMO site is where?
-before the transmembrane region (C-terminal)
-SUMO site: VKKE (Val-Lys-Lys-Glu); SUMO is added here
Scs2 N-terminus
MSP = + a.a residues; interacts with FFAT motif
_____ is nuclear envelope receptor for Siz2 during mitosis.
Scs2
Scs2, Siz2 interaction in late mitosis
-Siz2 is phosphorylated & SUMOlyates Scs2
-Siz2 FFAT motif interacts with Scs2 MSP motif (+++)
-SUMO site of Siz2 adds SUMO to Scs2
end of mitosis: Scs2 remove ____; Siz2 _____
-SUMO
-dephosphorylated
Recruitment of Siz2 to the nuclear envelope coincides with the reestablishment of _____
chromatin interactions with the nuclear envelope
Siz2-mediated SUMOylation at the nuclear envelope facilitates the reassociation of ______
telomeres to the nuclear envelope during mitosis
____ is required for the association of telomeres with the nuclear
envelope during mitosis
-Sir4
Nuclear envelope recruitment of ____ and SUMOylation of ____ are required for telomere association with the nuclear envelope.
-Siz2
-Sir4
chromatin IP (CHIP)
-pull down chromatin to see proteins associated with DNA
In late mitosis, a cascade of _____ faciliates the re-assoaction of chromatin to nuclear envelope during mitosis + association of proteins associated with NE
SUMOlyation
