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CELL 301 > Part B: Lecture 6 + 7 + 8 > Flashcards

Part B: Lecture 6 + 7 + 8 Flashcards

1
Q

Nuclear envelope (NE) composed of ______. Membranes are fused at
numerous positions along the NE to form_____. Densely staining
heterochromatin interacts with ______. Euchromatin is positioned _____.

A

-two membranes, outer and inner
-pores
-the inner membrane
-adjacent to pores

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2
Q

Electron microgram: Dark stain = _______
Less stain = _______

A

-heterochromatin (usually transcriptionally silent). Typically along the periphery
-euchromatin (usually transcriptionally active)

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3
Q

Outer membrane of nucleus is continuous with ______.
Lamina is a layer of proteins made up of proteins called _____. Lamina is generally associated with _____.

A

-inner membrane and ER
-lamins
-heterochromatin

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4
Q

Outer membrane is biochemically very similar to the ___, but the inner membrane is quite different

A

-ER

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5
Q

Nuclear pore complexes are positioned in pores formed by _____

A

the fusion of the inner and outer nuclear membranes

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6
Q

The number of NPCs per nucleus varies in _____ and appears to correlate with the ____. Pores are not randomly distributed. Their interactions with ______ dictate how they’re organized.

A

-different cell types
-transcriptional state of the cells
-chromatin and certain genes

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7
Q

Transport through the nuclear pore complex occurs by two separate mechanisms: ______

A

1) By passive diffusion of molecules less than 9 nm in diameter. This includes ions and metabolites.
2) Most proteins and RNAs (including those less than 9 nm in diameter) are actively imported by a process that is signal-mediated and energy dependent -> only verrry small proteins diffuse through

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8
Q

NLS stands for ____ + descr.

A

nuclear localizing signal; + Arg/Lys residues; transport into nucleus

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9
Q

NES stands for ____ + descr.

A

nuclear export signal; hydrophobic residues; transport out of nucleus

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10
Q

snRNA stand for ____ + function. snRNPs? hnRNPs?

A

-small nuclear RNA
-small nuclear ribonucleoproteins (form splicesome for removing introns)
-heterogenous ribonucleoproteins (modification of premRNA)

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11
Q

Nuclear targeting sequences are what? where? function?

A

-The signal is a noncleavable targeting sequence.
-The signal can be located anywhere on a protein, but it must be exposed at some point.
-Distinct signals mediate nuclear import and export.
-Signal are recognized by the mobile phase of the transport machinery

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12
Q

Stationary phase of Nucleocytoplasmic Transport

A

Nuclear Pore Complexes (NPCs); gateway of nucleus

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13
Q

Mobile phase of Nucleocytoplasmic Transport

A

Transport cargoes and transport factors; recognizes nuclear targeting signals

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14
Q

NPC are composed of about _____ in both yeast and
humans. Total mass of the NPCs range from _____ depending on the species (about___ the mass of a ribosome).

A

-30 different proteins
-70 to 100 MD
-25x

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15
Q

Nuclear pore complexes are composed of proteins termed ____

A

-nucleoporins

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16
Q

Three types of nucleoporins

A

-FG nucleoporins
-non-FG nucleoporins
-integral pore proteins

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17
Q

Nucleoporins containing repetitive FG-containing peptides (Phenylalanine - Glycine) do what?

A

line the transport channel and function in binding transport factors
( i.e. the soluble phase of the transport machinery).

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18
Q

Nucleoporins lacking FG repeat peptides (Phenylalanine - Glycine) do what?

A

form the scaffold of the NPC
-they also play an important role in NPC assembly and the organization of the FG-nucleoporins in the transport channel

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19
Q

Nucleoporins that are integral pore membrane proteins do what?

A

-also contribute to the NPC scaffold
-they contribute to anchoring the NPC to the pore membrane

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20
Q

problems with cyroEM

A

We don’t have much structural information about the proteins inside the hole because cryoEM looks at thousands of pores that overlap.
-better structural resolution when things are in a fixed position
-when things are not in a fixed position so they look different from every view, which makes it hard to see the actual structure

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21
Q

The S. cerevisiae Kap-beta family

A

-Substrate binding (anything with nuclear signal)
-NPC targeting (they bind the FGNups)
-14 putative members
-Composed of HEAT repeats
-~17% identity between members
-Most homologous at N-termini
- Many have mammalian homologues
-Importins and Exportins

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22
Q

Karyopherins bind _____

A

FG-Nups

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23
Q

Kap general steps

A

-Karyopherin + Cargo binds
-Kap/cargo complex binds to FG repeat-containing nucleoporins
-multiple binding and release steps are believed to facilitate translocation through the NPC
-RanGTP binds to improt Kap and triggers cargo release in nucleoplasm
-transport path surrounded by large numbers of katyopherin docking sites; most docking sites are found on both sides of NPC
-no mechanical or motor-driven gate

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24
Q

Models for physical state of FG-Nups in the NPC

A

-Selective phase - the porins might interact with each other to form a meshwork/hydrogel
-Virtual gate - they don’t interact with each other but seclude the channel by extending out

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25
Q

Even if the barrier is occupied by things, a _____ must be overcome for things to move through NPC. Free energy required to go from a _____.

A
  • fairly large energy barrier
    -free state to traveling through the channel
26
Q

A cell can a perform non-spontaneous reaction by _____. In this case it allows the energy barrier to be basically 0.

A

coupling it with a spontaneous reaction (which is energetically favourable and releases free energy).

27
Q

Karyopherins are able to interact with the barrier (______). They can partition through ____

A

-bind to FG-Nups and release energy
- the meshwork with their cargo

28
Q

Ran is a small ____ with two forms (bound to GTP or GDP). In the nucleus it’s bound to ____. When GTP binds to ____, there is a conformational change and the ______

A

-GTPase
-GTP
-karyopherin
-NLS-containing cargo is released into the nucleus

29
Q

Export karyopherins partition through the pores _______ . But binding to cargo is dependent on ______

A

-the same way as import ones (interaction with FG-nups)
-forming a trimeric complex with RAN-GTP.

30
Q

Overview of nuclear transport (export)

A

-RanGTP + Karyopherin + export cargo form complex
-complex enters pore
-Karyopherin + export cargo exports out of nucleus

31
Q

Overview of nuclear transport (import)

A

-import cargo + Karyopherin form complex
-complex enters pore
-Karyopherin + RanGTP binds in nucleus + releases import cargo

32
Q

Key to all this important directionality of nuclear transport depends on ______. Needs ____ binding to ran changes conformation of RAN. _____ binds to ran in the ____ bound form and activates its _____. ______ maintain the RAN gradient

A

-having a high RAN-GTP concentration in the nucleus
-RAN-GEF (makes GTP bound)
-RAN-GAP
-GTP
-intrinsic GTPase activity
-GEF and GAP

33
Q

Components of in vitro import assay

A

1) Import cargo: NLS-human serum albumin- FITC*
2) Soluble transport factors: Cytosol or purified factors
3) Import competent nuclei: Cell permeabilized with digitonin (makes holes in the plasma membrane but not the nuclear envelope membrane).
4) Source of energy: GTP or ATP

34
Q

Satterly paper summary

A

-Found the mechanisms used by influenza A virus to disrupt host cell mRNA export
-influenza A viral protein that inhibits the host cell immune response is NS1
-NS1 interacts and suppresses factors in host cell mRNA export
-suppression of mRNA exports facilitates influenza virus replication

35
Q

Influenza A virus replication cycle

A

-Negative strand RNA in membrane enclosed virus
-virus enters cells through coated pit with sialic acid receptor
-vesicle is pH 5-6 -> endosome -> pH dependent fusion with parental nucleocapsid
-Negative strand is replicated into a positive strand, which is used to make more negative strands (which are repackaged to make more virus particles)
-packaging is made in ER and golgi apparatus then added to membrane

36
Q

The ____ protein of influenza A virus is a major virulence factor that is essential for pathogenesis. Its functions?

A

-NS1
-to impair innate and adaptive immunity by inhibiting host signal transduction and gene expression

37
Q

We show here that NS1 forms an inhibitory complex with
_______ (4), which are key constituents of the mRNA export machinery that interact with both mRNAs and nucleoporins to direct ______

A

-NXF1/TAP, p15/NXT, Rae1/mrnp41, and E1B-AP5
-mRNAs through the nuclear pore complex

38
Q

Increased levels of ______ (3) revert the mRNA export blockage induced by NS1.

A

NXF1, p15, or Rae1

39
Q

Furthermore, influenza virus. down-regulates _____, a nucleoporin that is a docking site for mRNA export factors. Because _____ are induced by interferons, downregulation of this pathway is likely a viral strategy to promote viral replication.

A

-Nup98
-Nup98 and Rae1

40
Q

pull down assay aka ______

A

immunoprecipitation

41
Q

GST-tags and the GST pull-down assay

A

-GST (Glutathione S-transferase) added to protein of interest to purify it by inserting GST DNA coding sequence next to code for protein of interest to make fusion protein
-GST has a strong binding affinity for GSH (Glutathione); beads coated with GSH
-the protein of interest attached to the GST will stick to the beads, isolating the protein from the rest of those in solution
-the beads are recovered and washed with free GSH to detach the protein of interest from the beads, resulting in a purified protein
-drawback of this assay: the protein of interest is attached to GST alters its native state

42
Q

NXF1 is mammalian form of _____

A

-Mec67

43
Q

P15 binds to _____ of NS1. E1B-AP5?

A

-C-term but not N-term
-N-term

44
Q

______ binds to DNA. As cells die, they pick up more of this in their nuclei. Use this to measure the ______.

A

-Ethidium Bromide
-percentage of cell death

45
Q

HIV is an ______. Pre-initiation complex must be imported into the ____. _______ at the nuclear pore favours HIV replication

A

-enveloped virus with an RNA genome
-nucleus and integrated into the host cell genome
-Chromatin organization

46
Q

Lelek paper summary

A

-Tested if NPC facilitates the targeting of HIV integration by acting on chromatin topology

47
Q

If you deplete Nup153, you also lose ____ (since ______). If you deplete TPR, ____ is not affected

A

-TPR
-Nup153 anchors TPR to the pores
-Nup153

48
Q

MOI aka _____

A

multiplicity of infection (number of infectious viruses added divided by the number of cells)

49
Q

HIV infection lead to depletion of ____

A

Tpr

50
Q

shRNA do what?

A

silence target gene expression via RNA interference

51
Q

Less _____ being made in TPR knockdown (in HIV)

A

viral RNAs

52
Q

In cells expressing HA-LEDGF/p75 they observed an increase of ____ fluorescence near the NE suggesting that _____ underneath the NPC. When _____ is overexpressed, Tpr seems more stabilized at the NE. _____ acts indirectly by anchoring Tpr to the NPC, while Tpr may have a more active role on _____ and ____ infection.

A

-GFP-Tpr
-LEDGF/p75 stabilizes Tpr
-Nup153
-Nup153
-LEDGF/p75
-HIV-1

53
Q

When TPR is overexpressed, it _____

A

draws a host protein that helps with viral replication to the nucleus

54
Q

histone modification H3K36me3 is associated with _____

A

actively transcribed genes

55
Q

histone modification H3K4me3 is associated with _____

A

regulatory regions

56
Q

HIV virus imbeddes genes in _____ in WT cells; ______ in Tpr KO cells

A

-transcriptionally active regions
-inactive regions

57
Q

The structure of chromatin around NPCs and the function of TPR are taken advantage of the virus to _______

A

-integrate into transcriptionally active parts of the genome, which facilitates its replication

58
Q

Depletion of TPR abolishes _____

A

euchromatin channels adjacent to NPC

59
Q

_____ is not required for HIV nuclear import but ____ is essential

A

-integrity of nuclear side of NPC (made of Tpr)
-Nup153

60
Q

depletion of Tpr reduces ______ but not ______

A

-HIV infectivity
-level of integration

61
Q

_____, which promotes viral integration into active genes, stabilizes ____ at the nuclear periphery and vice versa

A

-LEDGF/p75
-Tpr

62
Q

_____ participates in HIV-1 nuclear import independently of the integrity of the nuclear basket

A

-Nup153

CELL 301 (7 decks)

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