cell bio 6c
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what is the outer nucleus membrane continuous with?
outer: rough ER
what is the order of the nuclear membrane layers?
outer membrane, perinuclear space, inner membrane, nuclear lamina, chromatin
what are nuclear lamins?
- a discrete class of intermediate filament that forms a mesh like network associated with the inner nuclear membrane
- a reticulated filament system that provide structural support for the nucleus on the inside of the nuclear envelope
what regulates the assembly/disassembly of the network during the cell cycle?
what are major proteins in nucleus called?
nuclear lamins; individual lamins form filaments in plasma membrane
what kind of proteins are lamin?
what is the job of integral proteins?
to attach nucleus to cytoskeleton so nucleus is anchored in place (associate chromatin to this large nuclear envelope complex
What are laminopathies?
regarding mutations in genes encoding proteins of the nuclear lamina
What are the mutations in the nuclear envelope associated with?
muscular and premature diseases such as progeria
how many nuclear pores are in the nucleus?
what sequences are made in the nucleus but has to get out of the nucleus to function?
mRNA, tRNA, ribosomal RNA
during the cell cycle, what happens to the nuclear envelope?
breaks down into vesicles, which contain nuclear pores, which stay intact. Vesicles form around chromosomes and fuse to form membrane again
what is the rate of diffusion for nuclear pore?
1000 macromolecules per second of tRNA, ribosomal subunites, mRNPs
what is the purpose of having different domains in nucleoporins?
contain different functions, antibodies to nucleoporins block transport
what kind of proteins diffuse freely through pres?
- less than 60,000 molecular weight; larger molecules require carrier proteins and energy from GTP hydrolysis to pass through the pores
- pores can expand to diameter of about 26nm
can how we test that things with NLS will be moved to the nucleus?
transfer of nucleoplasmin absorbed to gold particles
who is gunter blobel?
won nobel prize in physiology r medicine for the discovery that proteins have intrinsic signals that govern their transport and localization in the cell
what are typical NLS signals like?
nuclear localization signals have several basic amino acids in a linear sequence
what is the role of importin?
NLS sequences binds to importin; single importin transports multiple proteins
how would you test for NLS signal?
clone t-antigen, cut, splice and paste on this t-antigen a stretch of basic amino acids (fluorescent). express it in a cell
what does it mean when yeast importin can be used in humans?
proteins that recognize NLS are highly conserved
what is importin?
nuclear transport receptors administrative assistant and recognizes domain and brings protein through nuclear pore
what does it mean that importins are recycled?
one importin can be used for multiple proteins
what is the role of ionophores in Ca release?
creates pores in plasma membrane or smooth ER, which allows calcium to flow out of sarcoplasmic reticulum and into cytoplasm, so we can change Ca levels using this ionophore
what happens when calcium level is raised in the cytoplasm?
GFP (fluoresecent) NF-AT fusion moves into nucleus; transcription factor turns genes on to be regulated
what are the steps of nuclear import?
- importin binds cargo, moves in, protein that bind GTP recognizes importin and binds, causes release of cargo inside nucleus
- b/c of GTP gradient (high on inside), GTP (Ran)-importin complex moves out of nuclear envelope.
- GTP is hydrolyzed and releases phosphate, thus release of RAN
- importin can now be reused
what are exchange factors?
proteins that regulate RAN for import/export; Ran in turn regulates export/import molecules. exchange factors exchange GTP for GDP and vice versa
how are the roles of exportin and importin correlated?
exportin helps importin get out of the nucleus when its bound to ran GTP
what does the mitochondria use in division?
Dynamin GTPase: responsible for endocytosis in eukaryotic cell and involved in scission of newly formed vesicles
what is special about the outer and inner membrane of the mitochondria?
no ribosomes on surface and inner membrane is folded in cristae
what are mitochondria and chloroplasts derived from?
ancient prokaryotic anaerobic symbionts (over 1500 millions year ago)
what does the mitochondria genome encode?
maintain small prokaryote like genomes encoding rRNAs, tRNAs, some replicative enzymes and subunits of membrane respiratory chain complexes
where does 98% of mitochondria proteins' genome reside?
what is different about prokaryote genes?
- they don't have introns or exons and they are polysystronic (a single mRNA encoding several different polypeptide chains)
- genomes of mitochondria are like genomes of prokaryotes
what makes up the genome of mitochondria?
humans have 17000bp in their mitochondria which encodes for 13 proteins, 2rRNAs, 22tRNAs circular genome
what are the porins in the outer membrane composed of?
protein complexes involved in generation of proton gradient ATP driven motor generate F-type ATPase same structure are formed by inner membrane of bacteria (evolutionarily conserved)
what are the sources of mitochondria protein?
600 proteins destined to function in mitochondria (some are synthesized in cytoplasm and imported into mito with NLS, others are from mitochondrial genes on mito DNA)
where does the synthesized polypeptide go with C-terminal PTS signal?
where does the target signal go after protein is transported to target?
cleaved off and protein folds in target compartment
what are polypeptides synthesized with for targetting in the chloroplast?
synthesized with chloroplast transit sequence (cleaved before folding in stroma) and/or secondary targeting sequence (directs protein to final location)
what is the function of TOM and TIM?
Tom is on outer membrane of mito and Tim is on inner membrane, they get together to create pore and bring unfolded protein into mito
where does the protein reside in the mitochondria?
membranes or matrix of mitochondria
what is the role of sec translocon?
translocates proteins synthesized on the rough ER into the membrane or lumen
what is the role of Toc and Tic?
translocate proteins into the membranes or stroma of chloroplasts
What is the role of sec and tat translocons?
translocate proteins into the thylakoid membrane (stacked) or lumen
what are the machineries for importing proteins into mito?
signal presequences, Tom-Tim, translocons, signal peptidase, Hsp70 chaperone, energy source from electrical potential and ATPase chaperone
what does TOM and TIM stand for?
Translocase of Outer/Inner Mitochondrial Membrane; they are made up of multiple polypeptides
what is the function of chaperones in the mito?
function on outside and inside of mito; functions to unfold on outside and pull protein through and fold correctly on the inside
describe the synthesis of mito protein
- synthesized on cytoplasmic free ribosomes
- bind to Hsp70 for delivery to TOM
- signal presequences of 10-70 basic and hydrophic amino aicds, usually at N-Terminus
- interactions with TOM20, 22 at first
- then with TOM 40
- unfolded to transit
- then interact with TIm 23, Tim17, and Tim 50
- hsp70 fold protein and pull it throug
- signal is cut off by signal peptidase
what are the two flaws of the synthesis of mitochondria movie?
- signal peptidase sits under Tim and cleaves sequence as soon as it arrives
- Hsp70 is also docking there to help fold protein
- signal has affinity for Hsp70
- TOM and TIM complex is the contact site where the 2 membranes are close together
what allows TOM and TIM to live in the membrane?
they have hydrophobic stretches in the genome
what Tim translocon is responsible for bringing protein into inner membrane?
how does the ADP/ATP antiporter enter the inner membrane?
- recognition of signal, translocation to intermembranous space and association with chaperones
- translocation from TIm22 to inner membrane
describe the bacterial protein export?
- generally use Sec translocon
- homologous translocon to eukaryote ER
- cleavable sec N-terminal signal sequences (25 basic and hydrophobic)
- translocate unfolded due to chaperone
- translocation require ATPase
- some proteins translocation via SRP (signal recognition particle)
- some are independent of Sec pathway
How does one separate matrix, outer and inner membrane?
- in medium of low osmolarity, mitochondrion swells and burst (inner membrane remains in tact), centrifugation leaves contents of intermembrane space in the nonsedimenting fraction, transfer to high osmolarity, which causes shrinkage of the matrix,
- density gradient centrifugation separates the outer membrane from the dense matrix
- disruption and centrifugation separate inner membrane from matrix components
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