what % of proteins go through the secretory pathway versus stay in the cytosol and are targeted elsewhere? where else might they be targeted?
30% of proteins are handled by secretory pathway at the ER
70% stay in cytosol or are targeted to mitochondria, nucleus, or peroxisomes
what % of liver volume do mitochondria comprise?
size of mitochondria?
0.5-1 micron diameter
function of mitochondria?
generate ATP to be used as cellular source of energy
important structural features of mitochondria?
outer mitochondrial membrane, inner mitochondrial membrane with cristae forming big folds, membrane space in between the 2 membranes, mitochondrial matrix in middle of it all
structure(s) of outer mitochondrial membrane?
porous to molecules up to 5-10 kDa
contains a protein translocation apparatus, TOM
structure(s) of inner mitochondrial membrane?
inner membrane is 70% protein, is impermeable even to protons
inner membrane is highly invaginated into cristae to increase surface area (cristae are variable)
has TIM complex for transport of peptides across it
membrane potential across inner membrane
what is within the inner membrane, re: proteins?
protein complexes of the electron transport chain and ATP synthase complex that catalyzes formation of ATP from ADP
what creates membrane potential across inner membrane?
matrix (inner) is negatively charged; inter membrane space (out) is highly positively charged
protons moving into the intermembrane space creates membrane potential
thus inner membrane is sealed tight to protons
makes it hard to get proteins into the mitochodria
what's different between mitochondria in the skeletal muscle vs. liver?
muscles have lots of cristae; liver do not have much cristae
b/c liver is more involved in metabolism, and muscle is dedicated to ATP production
what to mitochondrion do in sperm?
in flagella - so sperm can swim
what is lodged w/in the inner mitochondria membrane in terms of proteins and molecules?
channel proteins for translocation of metabolites:
pyruvate, malate, acyl-CoA, amino acids
what is in the intermembrane space?
between the 2 membranes
contains enzymes that phosphorylate other nucleotides apart from ADP, eg nucleoside diphosphate kinase, which converts GDP -> GTP
what is in the mitochondrial matrix?
1) hundreds of enzymes, including those for oxidation of pyruvate, fatty acids, ketone bodies to acetyl-CoA
2) enzymes that catalyze amino acid oxidation
3) enzymes of tricarboxyic acid cycle and urea cycle
4) mitochondrial genome, ribosomes, tRNAs, molecular chaperones for folding newly synthesized & newly imported proteins
what is the structure of the mitochondrial genome?
encodes 13 proteins
2-50 copies of genome per mitochondria, so up to 1000/cell
what does mitocondrial genome encode?
tRNAs and rRNAs for its own ribosomes
mitochondrial genes are txn into mRNA and tln in matrix on mitochondrial ribosomes
what proteins does the mitochondrial genome encode?
subunits for several components of respiratory chain, including cytochrome c oxidase, NADH dehydrogenase, apocytochrome b
genes for protein synthesis within matrix: 22 tRNAs, 12S and 16S rRNA for mitochondrial ribosomes
can gene migration occur btwn the universal and mitochondrial genomes?
b/c mitochondrial genome has different genetic code than universal genetic code
i.e. UGA is universal STOP codon; it is Tryptophan in mitochondria
AGG is R in universal code, STOP in mitochondrial genome
how do mitochondria come to exist?
they aren't assembled; divide into 2 from 1 existing mitochondria
how are mitochondria inherited in mammals?
what is evidence of mitochondria's prokaryotic origin?
endosymbiotic origin from prokaryotes, like chloroplasts in plants
evidence: prokaryotic character of irbosomes, molecular chaperones, and circular genome
where do most mitochondrial proteins come from?
synthesized on cytosolic ribosomes before targeting to outer membraen and post-translational import
what is the nature of mitochondrial targeting sequence?
15-35 residuce N-terminal sequence of positive, basic amino acids
it is cleaved in matrix by an endoprotease
also has a non-cleaved internal targeting sequence
when do proteins destined for mitochondria get transported?
what is the state of the peptide?
what brings peptide to mitochondria? what does it do?
important b/c protein is imported in unfolded conformation, which Hsp70 maintains
describe how peptide enters mitochondria once it is chaperoned there from the ribosome
1) Hsp-70 bound peptide binds to receptor on outer mitochondrial membrane; is presented to TOM complex; enter through aqueous TOM40 translocation channel
no energy involved
2) positively charged pre-sequence of peptide opens TIM complex on inner mitochondrial membrane
electrophoretic effect occurs, b/c targeting sequence is positive compared to inner part of matrix
TIM opens enough for peptide to get in through inner mitochondrial membrane
3) once in matrix, mitochondrial version of Hsp70 binds protein
4) Hsp70 pulls protein into matrix, helps fold it
what are all of Hsp70's functions?
1) keep protein unfolded in cytosol
2) pull protein in to mitochondrial matrix after presequence has entered through TIM complex of inner mitochondrial membrane
3) helps protein fold after it has entered matrix
when does the mitochondrial targeting sequence get cleaved?
once peptide is within the matrix
what happens if have a mutation in mitochondrial DNA?
may have genotypic mutation, but it may not express disease phenotype (although it may!)
penetrance determines this - more copies of a mutation you have in genotype, more experiences in phenotype
what is leber's hereditary optic neuropathy (LHON) caused by?
missense mutation in subunit 4 of NADH-coQ reductase
causes sudden onset blindness, hearing loss, mental retardation
what causes dystonia?
NADH-coQ reductase missense mutation in subunit 4
same mutation as LHON
what does aging due to mitochondria DNA?
rearranges mitochondrial DNA
probably result of accumulation of reactive oxygen species with time
humans over age 40 have increased mitochondrial DNA rearrangements compared to humans under age 40
leads to decreased energy production w/ age
what is the structure of peroxisomes?
small, single membrane organelle
what do peroxisomes do?
fatty acid B-oxidation aka they oxidize long chain fatty acids
this generates hydrogen peroxide which catalase reduces to water and oxygen
do plasmalogen synthesis - phospholipids found in omst eukaryotic cells, myelin membranes
what encodes peroxisomal proteins? where are they synthesized? when are they transported? how?
synthesized on cytosolic ribosomes
imported post-translationally in folded state into peroxisome
2 C-terminal target sequences: PTS1, it is SKL a.as
what does catalse do?
reduces hydrogen peroxide to water and molecular oxygen
works in peroxisomes
what are diseases related to peroxisomes? what are they caused by?
1) zellwegers syndrome - no import of any peroxisomal enzyme
2) adrenoleukodystrophy (ALD) - oxidation of very long chain fatty acids is defective; ALD gene is membrane transporter for long-chain fatty acyl CoA synthase from cytosol to peroxisome matrix
what does nucleus separate?
genetic material inside, separate from cytosol
separates txn and tln, allowing another level of regulation compared w/ prokaryotes
what activites occur inside nucleus?
when does nucleus form?
nucleus is disassembled prior to mitosis, reforms afterward
what is size of nucleus?
largest organelle in cell
5-10 um diameter
what is membrane of nucleus like?
double lipied bilayer
inner = nuclear lamina, gives nucleus tensile strength
outer layer = rough ER membrane, they are contiguous
what/where is nuclear lamina?
lines inner surface of nuclear membrane
meshwork of intermediate filament type proteins, lamins
how does transport into/out of nucleus occur?
via aqueous pores, nuclear pore complexes
what is the organization of chromatin?
nucleus houses all all genes, in 46 chromosomes
each chromosome is very long and thin; presents packing problem
DNA is packaged into chromatin which is further compacted into a 30 nm fiber
these 30 nm fiber are interphase nucleus chromatin structure
what is the nucleolus?
where rRNA is transcribed, synthesized
where ribosomal subunits are assembled
most prominent strucutre within nucleus
describe the chormatin of the interphase nucleus
euchromatin (10% of which is transcribed) and heterochromatin, which is highly condensed and not trnscribed
highly condensed chromatin is in close apposition to the lamina
euchromatin and heterochromatin define the interphase nucleus
during mitosis, chromosomes condense into metaphse chromosomes that're more highly organized
where are the genes that encode rRNA?
in nucleolus, 200 copies of these genes, including 5.8S, 18S, 28S on 5 different chromosomes
all are synthesized as a 45S precursor
these locii localize to the nucleolus
describe process of ribosomal RNA biogenesis
they congregate in the nucleolus of the nucleus
ribosomal mRNA are synthesized on chromsomes all around the nucleus by RNA Pol II
the mRNA are exported from nucleus; translated on cytplasmic ribosomes
ribosomal proteins then reenter nucleus, are transported to nucleolus, as does 5S RNA
there, assemble w/ rRNA
individual 40S and 60S pre-ribosomal aprticles are then delivered to cytosol
how many protein molecules enter nucleus / minute?
how many mRNA?
all via 3,000 nuclear pore complexes
what is the nuclear localization sequence?
can exist anywhere on protein
usually 4-8 a.as, rich in Arg and Lys, usually contains Pro
in what conformation do proteins enter nucleus?
are nuclear export signals = NLS?
nuclear export signal is distinct from nuclear localization sequence
when/is the nuclear localization cleaved?
because proteins shuttle between nucleus & cytoplasm or have to reenter nucleus as it reforms after mitosis
how do proteins enter/exit nucleus?
through nuclear pore complexes: 8 subunits long ring
bidirectional transport occurs through NPCs
what is structure of the NPC?
nuclear pore complex
3000-4000 per nucleus
each composed of ~100 diff proteins, collective mass is 125 Md
has an 8 fold symmetry, contains central aqueous channel of 9 nm diameter and 15 nm length
is several ring assemblies that occupy cytoplasmic face, inner core and nucleoplasmic face of structure
filamens radiate out from both cytoplasmic and nucleoplasmic sides
describe process of protein entry into the nucleus via the NPC
if protein is made in cytosol, wants to go back to nucleus to ribosome
1) protein binds to karyopherin receptor
karyopherin enters through NPC w/ protein attached
2) RAN-GTP causes disassembly of karyopherin complex, cargo protein is released
3) karyopherin is bound to RAN-GTP, they undergo export through NPC back to cytosol
4) cytosolic RAN-GAP protein promotes disassembly of karyopherin:RAN complex by catalyzing GTP hydrolysis in Ran
karyopherin now free to recycle, pick up new cargo; Ran GDP can return into nucleus, and Ran ntd exchange factor, RAN Nef, catalyzes formation of Ran GTP to start cycle again
what is Ran? what are its forms/where? relations to GAP and GEF?
a small GTPase w/ co-factors that regulate nucleotide hydrolysis (Ran GAP) and ntd exchange (Ran GEF)
Ran GAP: cytosol, so Ran GDP
Ran GEF: nuclear, so Ran GTP
during process of secretion:
is there a signal seuqnce? is signal sequence cleavable? co or post translational?
yes, signal sequence
during transport into mitochondria,
is signal sequence cleavable?
co or post translational?
re: transport to nucleus,
is signal sequence cleavable? co or post translational?
is signal sequence cleavable?
co or porst translational?
not cleavable, and at C terminus