Part B: Lecture 6 + 7 + 8 Flashcards by Tami Ojewole

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

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

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Electron microgram: Dark stain = _______
Less stain = _______

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

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

-inner membrane and ER
-lamins
-heterochromatin

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

-ER

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Nuclear pore complexes are positioned in pores formed by _____

the fusion of the inner and outer nuclear membranes

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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.

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

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

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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NLS stands for ____ + descr.

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

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NES stands for ____ + descr.

nuclear export signal; hydrophobic residues; transport out of nucleus

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snRNA stand for ____ + function. snRNPs? hnRNPs?

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

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Nuclear targeting sequences are what? where? function?

-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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Stationary phase of Nucleocytoplasmic Transport

Nuclear Pore Complexes (NPCs); gateway of nucleus

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Mobile phase of Nucleocytoplasmic Transport

Transport cargoes and transport factors; recognizes nuclear targeting signals

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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).

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

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Nuclear pore complexes are composed of proteins termed ____

-nucleoporins

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Three types of nucleoporins

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

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

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

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

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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Nucleoporins that are integral pore membrane proteins do what?

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

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problems with cyroEM

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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The S. cerevisiae Kap-beta family

-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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Karyopherins bind _____

FG-Nups

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Kap general steps

-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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Models for physical state of FG-Nups in the NPC

-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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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 _____.

- fairly large energy barrier -free state to traveling through the channel

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

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

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

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

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 ______

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

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

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

Overview of nuclear transport (export)

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

Overview of nuclear transport (import)

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

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

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

Components of in vitro import assay

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

Satterly paper summary

-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

Influenza A virus replication cycle

-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

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

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

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 ______

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

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

NXF1, p15, or Rae1

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.

-Nup98 -Nup98 and Rae1

pull down assay aka ______

immunoprecipitation

GST-tags and the GST pull-down assay

-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

NXF1 is mammalian form of _____

-Mec67

P15 binds to _____ of NS1. E1B-AP5?

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

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

-Ethidium Bromide -percentage of cell death

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

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

Lelek paper summary

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

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

-TPR -Nup153 anchors TPR to the pores -Nup153

MOI aka _____

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

HIV infection lead to depletion of ____

Tpr

shRNA do what?

silence target gene expression via RNA interference

Less _____ being made in TPR knockdown (in HIV)

viral RNAs

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.

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

When TPR is overexpressed, it _____

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

histone modification H3K36me3 is associated with _____

actively transcribed genes

histone modification H3K4me3 is associated with _____

regulatory regions

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

-transcriptionally active regions -inactive regions

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

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

Depletion of TPR abolishes _____

euchromatin channels adjacent to NPC

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

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

depletion of Tpr reduces ______ but not ______

-HIV infectivity -level of integration

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

-LEDGF/p75 -Tpr

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

-Nup153

Part B: Lecture 6 + 7 + 8 Flashcards

(62 cards)