Cytosol Nucleus Tracking Flashcards
Slide 4
Scanning EM image of nucleus nuclear envelope, cytoplasm
What is the perinuclear space with?
Endoplasmic Reticulum
How many nuclear membranes are there?
“2” - outer & inner!
What else is in the nuclear envelope?
Pore complexes!
Fibrous Lamina (or Nuclear Lamina)
A meshwork of intermediate filament proteins lining the inner aspect of the inner nuclear membrane
Which materials are exchanged between the nuclear and cytoplasmic compartments?
Slide 6 Nuclear proteins (in) Ribosomal proteins (in) Ribosomal subunits (out) mRNA (out) tRNA (out) Shuttling proteins (out) Ions (both) ATP (both) Smallecules (
Nuclear Envelope Components (Start presentation here?)
Nuclear Membranes
Nuclear Lamina
Nuclear Pore Complex
Slide 10 is helpful
Outer Nuclear Membrane
Continuation of RER, has ribosomes on its outer surface
Inner Nuclear Membrane
Associated with Nuclear Lamina & Chromatin
Pore Membranes
Connect the two!
Associated with pore complexes!
Nuclear Lamina
Slide 11 & 12
Made of 10 nm-diameter fibers (characteristic of IFs)
Components of a nuclear lamin IF
Variable Head & Tail Domains
Highly Conserved alpha-helical rod domains (responsible for dimerization), then higher polymerization into IFs
What makes Lamin IFs different from cytoplasmic IFs
They have more repeats in the rod domains
They have NLS
What happens to the nuclear lamina during mitosis?
The lamina proteins get phosphorylated, causing them to disassemble into free-floating dimers.
They later re-assemble by being dephosphorylated.
What proteins does the lamina interact with in the inner nuclear membrane?
LBR Emerin POM121 gp210 Myne-1 Nurim Nuclear Pore Complex MAN1 LAPs HETEROCHROMATIN 60 - 80 proteins, total
What are the two types of lamins that make up the Nuclear Lamina?
A-Type
B-Type
Nuclear Pore Complex
Slide 15
Slide 16 - Pretty!!!! (dissolved membrane away with detergent, but the pore complexes remain)
Nuclear Pore Complex Components
Slide 17!!!! Cytoplasmic Fibrils Cytoplasmic Ring Nuclear Ring Nuclear Basket Distal Ring
What proteins make up a Nuclear Pore Complex?
Nucleoporins (many have FG repeats)
Integral Membrane Proteins (gp210, POM121)
Associated Proteins (Ran, Lamins)
Others not well characterized
Mechanisms of Nuclear Transport
Diffusion
Active Nuclear Transport
Diffusion
Chemical and electrical gradient equilibrium
Works in NPC for smallecules like ions and ATP
Limited by permeability of membrane and diameter of pores
Rate of Diffusion
Inversely proportional to square root of the molecular mass. Some molecules (like histones) can diffuse, but not as quickly as you'd like, so there are alternative mechanisms.
Diffusion Size Cutoffs
60 kDa and above can’t diffuse into nucleus
Some molecules concentrate in the nucleus, even though they could freely diffuse. Why?!
They bind to thangs in the nucleus. Ex: Cations that bind to negatively-charged DNA
Active Nuclear Transport
Faster uptake for larger molecules that could diffuse, but slowly.
Uptake of molecules to large to diffuse
Requires ATP or GTP
NLS code
PKKKRKV
Pro-Lys-Lys-Lys-Arg-Lys-Val
KKKRK is the most important
Mess up something in there, and the NLS won’t werq
Discovered from the SV40 Large T Antigen (viral)
Also, hella basic
What happens when you covalently attach to albumin or gold?
GOLD ASS NUCLEUS
A few examples of NLS sequences
PKKKRKV GKKRSKA KAKRQR RKRRR PRRRK PYLNKRKGKP KKKKRKREK KKKRRSREK
Second type of NLS
Bipartite (discovered in Nucleoplasm)
KR PAATKKAGQA KKKK
Two basic clusters separated by a sequence of about 10 rando amino acids
Two step process of nuclear import
Step 1 - Energy Independent
Step 2 - ATP Dependent
Step 1
Fraction A
Binding of NLS-containing substrate to to a nuclear pore complex
NEM-sensitive
Step 2
Fraction B
Translocation - Moving of the substrate across the pore.
Blocked by non-hydrolyzable GTP analogues
Fraction B
Contains Ran (Ras-like protein that binds GTP)
Restored import in the presence of ATP and Fraction A.
Recombinant Ran also active.
Blocked by non-hydrolyzable GTP analogues
What does this mean?
It was primarily Ran-dependent hydrolysis of GTP that provides energy for transport across the pores
Fraction A
Heterodimeric complex needed for NLS-mediated import Karyopherin alpha/Karyopherin beta Importin alpha/importin beta NLS receptor/p97 NPC targeting complex
THESE ARE ALL NAMES FOR THE SAME COMPLEX
Typically karyopherin or importin are the running names these days.
Karyopherin/Importin Alpha - Important structure
N-terminal, C-terminal, IT IS A PROTEIN
Functional shit:
NLS Binding/Contact Surface
Karyopherin/Importin Beta1 binding domain
Karyopherin/Importin Beta1 - Important structure
The first of a family of karyopherinBetas/ImportinBetas
Involved in SV40 Large T antigen and Bipartite NLS
90 kDa
Binds importin-alpha (via importin beta binding domain)
Binds RAN-GTP
Binds FG repeats in nucleoporins
There is a large family of these proteins. Some mediate import. Some mediate export.
Beta1 needs Alpha (middleman) to bind to the NLS.
Most other karyopherin Betas bind directly to cargo.
They bind to:
Ribosomal proteins
tRNA
Some are involved in export, but rely on an NES for that.
NES
Nuclear Export Sequence
Very Leucine-rich.
The tRNA one appears on the tRNA itself, not a bound protein
“Important things” about Karyopherin/importin(exportin)-Betas
Over 20 in the human genome
Each receptor recognizes a different type of targeting signal for import or export
Soluble
Interact with both Cargo (sometimes indirectly) and NPC
Shuttle back and forth through NPC carrying cargo one way, then recycling back without cargo (usually)
mRNA export
Not completely understood.
Coupled to splicing somehow.
Inhibit splicing, inhibit export.
No karyopherin/importin involved.
Ran-GAP
Activates the cytoplasmic GTPase so Ran-GDP predominates in cytoplasm
Ran-GEF
Catalyzes exchange of GDP for GTP.
Importins release their cargo when?
Upon binding Ran-GTP
Exportins bind their cargo when?
When they bind to Ran-GTP
Exportins release their cargo when?
When Ran-GTP is no longer around.
NLS and NES signals can be masked.
Phosphorylation - conformational change
Phosphorylation - binding to another protein that hides the signal
NLS and NES signals can be created
Phosphorylation - Conformational change
Phosphorylation - Release fo masking protein
Degradation of bound masking protein
What does the nuclear envelope do besides all this?
Regulates transcription factors through MAN1 and LEMD3
MAN1/LEMD3
Brake
Resides in inner nuclear membrane
Downregulates Smad 1/2/3
Smads 1/2/3
Move into the nucleus (using karyopherins) in response to activation of the TGF-Beta signaling family
What happens if you lose half your MAN1?
Sclerosing Bone Dysplasias with Skin Abnormalities.
Buschke-Ollendorff Syndrome and Osteopoikilosis
From increased TGF-Beta and BMP signaling in bone and skin
Osteopoikilosis and Buschke-Ollendorff
Hyperdensities in cortical bone (can be painful)
From overactive BMP and TGF-Beta laying down the cortical bone.
Also has skin effects too.
Last slide
Um I don’t think it’s on the test.
memorize it maybe?
I think maybe it was just to make this all seem relevant.
