Exam 2b Flashcards
What do proteins define in each compartment of a cell?
function
where are localization signals in a protein
they are part of primary or secondary structure
nuclear localization signal (NLS)
directs proteins to nuclear pore complex (NPC)
what is in localization signal
proline followed by positive amino acids
where can localization sequence be
anywhere in protein
what size things can diffuse freely into nucleus
5kDa small molecules, dNTP, NTP, small proteins
what size things for sure need assistance getting into nucleus
30kDa, large proteins, RNAs
characteristics of nuclear porin
huge, has 30 proteins, many copies, approximately 120 Mda
what is NPC center like
gelatinous and disordered
how is NPC selective
for size
what is nuclear porin gate made of
glycine and phenylalanine, which are both nonpolar
key players in nuclear import/export
nuclear import receptor, nuclear export receptor, Ran, RanGTP, RanGDP, RanGAP, RanGEP
nuclear importer receptor
floating in cytosol, interacts with NLS, directs proteins to nuclear pore
Ran
monomeric G protein, binds receptor when bound to GTP in nucleus, shuttles import receptor back out
Ran-GAP
in cytosol, releases Ran from receptor GTP to GDP
Ran-GEF
in nucleus, converts Ran-GDP to Ran-GTP, maintains Ran-GTP
active and inactive form of Ran
Ran GTP is active/ Ran GDP is inactive
what limits NLS exposure
ligand binding/conformation
what do nuclear export receptors do
go into nucleus, pick up proteins with nuclear export signal and deliver it to cytosol
What role does Ran-GTP play in export
promotes cargo association
what role does Ran-GAP play in export
induces receptor to hydrolyze GTP to GDP. Then export receptor releases both cargo and GDP in cytosol, the returns to nucleus
how to test if sequence is enough to guide to organelle
bind to protein and see if it goes. Cleaving it may just ruin protein so it can’t go anywhere.
endoplasmic reticulum
extensive network of membrane/ expansive and dynamic
smooth ER
lipid and steroid synthesis
rough ER
protein synthesis for entire endomembrane system
signal peptide (SP)
for entry into ER or for secretion of bacterial proteins
where is signal peptide and what is it?
at the N-terminus, a hydrophobic alpha helix, approximately 15-30 amino acids
when do most proteins enter ER
during translation, “co-translational”, reason for RER formation
what happens during ER signal sequence
translocation
How does protein bind to ER
Er signal sequence on newly formed polypeptide chain binds to SRP, which directs the translating ribosome to the ER membrane. SRP binds to receptor! Receptor calls translocator. Dissociates from protein. Signal enter translocator. Protein on C end comes in and comes through. Signal is cleaved off in membrane.
pH of lumen ER
Approximately 6
ph outside lumen of ER
7
2 types of ribosomes
free ribosomes and membrane bound ribosomes (coat rough ER)
Type I transmembrane protein
has signal peptide and is equal to or greater than 1 hydrophobic alpha helix
which side of start-transfer protein ends up facing cytosol?
the more positive side stays out
Type II transmembrane protein
No SP, has “internal start transfer” sequence
Post-translation
fully synthesized,then directed into ER
what translocator do bacteria, archea and eukaryotes all have
Sec 61
What complex do eukaryotes use for translocation post-translation
Sec 62, 63, 71, 72 complex
what does sec 62, 63, 71, 72 complex do
which attaches itself to Sec 61 and deposits BIP molecules onto protein chain as it emerges through translocator.
How does BIP work
BIP is ATP driven. Release pull protein into lumen
sec Y
translocator complex used by bacteria
what feeds sec Y
Sec A ATPase with conformation changes that cause piston like motion in Sec A. only in bacteria.
two post translational modifications in ER
N-linked glycosylation; Disulfide bond formation
when does glycosylation happen? To what?
-as proteins enter or after;-both soluble and transmembrane proteins
sequence for attaching oligosaccharide
Asn-X-Ser/thr
how does N-glycosylation end
in three sugar residues
why n-glycosylation
increase solubility of protein; prevent proteolysis, can become part of glycocalyx if transmembrane, part of zip code for lysosome, keeps misfolded proteins in ER
how does it prevent proteolysis
protein can have many oligosaccharides attached to it, and they shield it from proteases
how are misfolded proteins kept in ER
misfolded proteins are held back; are recognized by glucose residues still attached to it; chaperone protein calnexin binds to glucose residues of protein; Protein is released from clanexin when glucosidase removes terminal glucose residues;Glucosyl transferase determines whether folded correctly or not;if it isn’t, the transferase adds new glucose from UDP-glucose
what happens if protein never folds correctly
;chaperones, disulfide isomerases and lectins are involved
-go to cytosol;ubiquitylated, deglycosylated and degraded in proteasome
how does n-glycosylation work
Oligosaccharide is attached to PM, neighboring a growing peptide chain. Oligosaccharide protein transferase transfers oligosaccharide to peptide chain.
what forms SS bond
protein disulfide isomerase or pdi;-helps correct disulfide bonds to form
There are no disulfides in cytosolic proteins. Why?
because of reducing environment, which means there is glutathione that breaks SS bonds
where do SS bonds exist
only inside organelles and extracellularly
what happens to SS bonds in cytosol
become SH sulfhydryl groups
what do sulfhydryl groups mean to ER
a signe of incomplete disulfide bond formation
can there be SS bonds in nucleus
no. it has a reducing environment like cytosol
What is the endomembrane system?
ER, Golgi (has cisternae), PM, endosomes, lysosomes/vacuoles, vesicles, peroxisomes
what is entry point to endomenbrane system
ER
what make EM a system
exchange of materials
what kind of transportation does EM rely on
vesicular transport
what is pH of ER
6
is lumen of EM acidic or basic
acidic
what is pH of lysosome. Why?
- due to H+ATPase, which adds protons
what happens to pH as proteins move farther into EM
gets more acidic
three parts of vesicular transport
formation (budding), movement to target membranes, fusion with target membranes
what are players in vesicle formation
cargo, receptor, adaptor, coat proteins, dynamin
vesicle formation steps
initiate bud, coat proteins drive vesicle formation, adaptors have clathrin triskeleon, then dynamin pinches off vesicle, coat falls off
what does dynamin use to pinch off vesicle
GTP; wraps around and pinches off
are adaptors always open
no, they are locked sometimes
with what do different adaptors associate
different membranes and different coat proteins via phosphoinositides in cytosolic leaflet
what can be modified in phosphoinositides
three OH groups after carbon 1
how are different PIPs produced
phosphorylation of 1, 2 or 3 carbons can form a variety of species
do animal cells only have one PI or PIP
no, they have various
what catalyzes PIP production
PIP phosphatase
where do PIPs live
in different membranes and different domains
what are PIPs associated with
specific vesicle transport events
vescicle membrane steps
Has PIP, fuses with PM, PI kinase adds P, recruits adaptor protein, initiate clathrin coated pit, vesicle hydrolizes and loses coat
how do coat proteins drive vesicle formation
via autoassembly
what is triskeleon made of
three heave clathrin chains and three light chains
types of coat proteins
clathrin, COPI, COPII
what do different coat proteins do
select different cargo
how does vesicle move
via cytoskeleton, does not float, motor proteins move vesicle along microtubule towards, for example, cis golgi
what does vesicle fusion with target membrane require
Rab and SNAREs
what form of Rab is active
Rab-GTP
what does Rab-GTP do
identifies target membrane
what do SNAREs do
drive vesicle fusion
how are SNARES built
have hydrophobic side and hydrophilic side extracellularly, so form coiled coils;have one transmembrane domain and one or two long amphipathic alpha helices
Coiled coils
nonpolar wrap around each other (two alpha helices, eg.);can join both in parallel and antiparallel direction
Types of SNAREs
v-SNARE –vesicular-attaches to vesicle;t-SNARE – target membrane – attaches to membrane
Vesicle fusion Steps
- Rab effectors does initial tether of vesicle to target membrane;2. the two different SNARE membranes pair;3. vesicle docks;4. fusion – Rab GAP hydrolyzes Rab GTP to Rab GDP, which then dissociates from membrane and returns to cytosol bound to GDI (keeps Rab soluble and inactive)
what happens to membrane orientation during fusion
;Membrane orientation is maintained with each budding and fusion event;one leaflet always faces cytosol
Botulism case
Man eats food contaminated with clostridium botulinum, which has toxin protease that cleaves SNAP25, t-SNARE no longer available, vesicle can’t bind to membrane in neuron terminal, so neurotransmitters can’t be released;With no vesicular fusion, there can be paralysis or death.
dissociation of SNARE pairs by NSF after fusion
;NSF binds to SNARE complex;has accessory proteins help; hydrolyzes ATP to pry SNAREs apart
membrane orientation during vesicular formation
-membrane orientation maintained with budding and fusion; that is, if C terminus is facing cytosol in organelle, it will face it in vesicle and in target membrane
charge of amino acid terminus
positive
golgi apparatus
– condensed stacks of membrane near cell center
golgi parts
cis golgi – receives from ER; trans golgi – stuff leaves golgi from here
major functions of golgi
- modification/synthesis of glygolipids and glycoproteins;2. golgi is post office
Secretory (anterograde) pathway
ER secretes vesicles vesicles form vesicular tubule cluster proteins marked with KDEL use retrograde pathway and return to ER other vesicles go to cis Golgi vesicles leave cis Golgi and go to cis, medial and trans Golgi cisternae vesicles go to trans Golgi vesicles leave trans Golgi and head to plasma membrane
The Golgi as post office
sorts to lysosome -sends proteins with mannose-6-phosphate (MSP) marker to lysosomes via endosomes; sorts to plasma membrane - -sends items to PM to be secreted or to become part of PM
Two types of secretion:
. constitutive and regulated
constitutive secretion
-all cells do constitutive exocytosis to maintain PM and extracellular space
regulated secretion
-signal mediated;-special, glandular cells send insulin, neurotransmitters, etc.;-dense vesicles aggregate and wait for signal so they can then cause short burst of a lot of protein
Exocytosis vs endocytosis in endomembrane system:
In growing, dividing cells : exo > endo;In nongrowing cells : exo = endo
Endomembrane system is:
- acidic ~ pH 5;2. has high Ca2+ concentration;3. causes soluble proteins to aggregate and form dense vesicles (not all proteins aggregate under acidic conditions)
Zip code for secreted proteins:
N-| SP | —————————— |-C; SP and nothing else – protein will be secreted from cell
golgi to lysosome zip code
Zip code: mannose-6-phosphate at N-glycosylation site ;N-| SP | ————Asn – X – Ser/Thr ————|-C;Has SP to get in ER;Golgi kinase uses ATP to ADP to phosphorylate sugar residue on protein (adds mannose-6-phosphate)
Trip to lysosome
ER Golgi endosome lysosome
Lysosome function
-recycling bin-acidic, pH ~ 5-H+ATPases – constantly pumps H+ into lysosomes-filled with digestive enzymes – acid hydrolases-has many exporters on surface
List of acid hydrolases:
Nucleases Proteases Glycosidases Lipases Phosphatases Sulfatases Phospholipases
how does protease in lysosome treat protein
Protease in lysosome takes protein, degrades to amino acids and allows amino acids to be used to build other proteins
Lysosomal Storage Disease:
-proteins don’t reach lysosomes-end up with overaccumulation-Inclusion cell disease – problems with transport of all hydrolytic enzymes to lyposomes-Tay-sachs – problems degrading glycolipids
Pinocytosis
-small stuff-means cell drinking-via vesicular trafficking-all eukaryotes-regulated vs constitutive
constitutive pinocytosis reason
to rejuvenate PM and extracellular space
regulated pinocytosis reason
for specific material, receptor mediated
regulated example in liver
Liver synthesizes LDL – bloodstream – LDL receptor – adaptin – clathrin coat – coat off – endosome – lysosome; From acidic endosome, receptor disassociates LDL and recycled back to PM; In lysosome, cholesterol is digested and secreted into cytosol
More on the endosome
-first place pinocytosed materials go-acidic, receptors release cargo, go back to PM-acts like trans Golgi Network (TGN) (sorting);receptors back to PM or other stuff to lysosomes
homotypic fusion
Endosomal fusion
where do digestive enzymes fuse with endosomes
TGN
what do endosomes have internally
multivesicular; have RNA
Phagocytosis =
-large stuff-cell eating-foreign cells, cell debris (digested in lysosome)
what does phagocytosis
white blood cells, amoebae, protists
Autophagy:
self eating-for damaged organelles or starvation conditions, especially mitochondria
Four degradation pathways
- from PM to phagosome to lysosome 2. from PM to phagosome to endosome to lysosome 3. from PM, endocytosed, early endosome, late endosome, lysosome 4. autophagy (double membraned autophagus)
Inner mito membrane characteristics
-has no cholesterol, has infolding to form cristae, has electron transport system and ATP synthase, has enzymes for lipid synthesis
why is inner membrane space more acidic than matrix space?
Protons are pumped into this space by the complexes in the ETC.
Outer membrane characteristics
-has cholesterol, contains import receptors and porins for import of large molecules
What encloses matrix compartment?
Inner membrane
What does matrix compartment contain?
The DNA and dense concentration of proteins including those for transcription and translation activities, and enzymatic reactions like the citric acid cycle and fatty acid oxidation.
What is inner membrane compartmentalized into? 2
Inner boundary membrane and crista membrane.
Three distinct spaces in the mitochondrium
Inner membrane space, crista space, and the matrix.
What is crista junction?
Joins cristae to inner boundary membrane
What led to an aerobic eukaryotic cell?
The uptake of a bacterial cell with ability for respiration
What was initial host cell of this bacterium? How did it gain a competitive advantage?
An anaerobic primitive eukaryotic cell or a type of archeal cell. With help of endosymbiont.
To where did genes from endosymbiont move? How may proteins do modern mitochondrial genomes code for?
To the nucleus. 13 proteins.
What is Rickettsia prowazekii?
An obligate intracellular parasitic bacterium that causes typhus.
What is important about R. prowazekii?
It provides clues about the origins of mitochondria. Its bacterial genome is most closely related to mitochondrial genomes. It has 834 open reading frames – ten times more than most mito genomes.
What gave rise to chloroplasts?
An endosymbiotic oxygen loving cyanobacterium (cyan)
From what did mitochondria arise?
Alpha-proteobacterium
What are the nearest relatives of mitochondria?
Three closely related groups of alpha-proteobacteria – the rhizobacteria, agrobacteria, and rickettsias.
What kind of photosynthesis did ancestral fermenting bacteria do?
H2S
What did ancestral fermenting bacteria evolve into?
Reducing atmosphere – green sulfur bacteria and purple sulfur bacteria. Oxidizing atmosphere – green filamentous bacteria, purple nonsulfur bacteria, and cyanobacteria.
What happened before the rise of cyanobacteria?
H2O photosynthesis
What three types of bacteria did purple nonsulfur bacteria break into?
Beta proteobacteria, alpha proteobacteria, and gamma proteobacteria
What is extranuclear inheritance?
Phenotype does not segregate with nuclear genes.
Yeast example of extranuclear inheritance
petite mutants grow very slowly and are deficient in respiration. The phenotype disappeared in cross of petite mutants to wild type strains. All diploid progeny were wild type. When these strains were sporulated, all haploid progeny were wild type. Poky phenotype did reappear sporadically as the haploid progeny continued to divide. They showed non-Mendelian inheritance.
How many mitochondria do cells contain?
Multiple
How many copies of DNA does each mitochondrion contain?
Multiple
What did yeast progeny cells contain mitochondria wise?
They were heteroplasmic – contained multiple mitochondria, some mutant and some wild type.
When did mutant yeast phenotype get expressed?
When WT mito fell into minority.
What was the name of the mutant Neurospora crassa fungus?
Poky
What is unusual about neurospora crassa crosses?
The females provide nucleus and cytoplasm. The males only provide nucleus. In crossing poky females to wt males, progeny were poky. If females were wt, progeny were wt.
What happened when cytoplasm from “poky” cells was injected into wild type cells?
The wild type recipients became “poky”. The mitochondria had the ability to induce the “poky” phenotype. The poky mito have a replicative advantage over wt mitochondria and quickly become the dominant mitochondria in the cell.
From where does a human inherit the mitochondria?
The mito DNA is inherited only from the mother’s egg. The sperm contributes a nucleus only.
What DNA do human mitochondria contain?
A circle 5 microns in length, containing 16 kb of DNA.
How is yeast mito DNA different from human?
It is five times larger, but has the same amount of genetic information.
What do mitochondrial genomes encode?
All the tRNAs and ribosomal RNAs necessary for translation in the mitochondria.
How many tRNA genes are there in mammalian mito?
22 tRNA genes
How long is human mito DNA? Yeast mito DNA?
5microM vs 25 microM
Are there introns in human mito DNA?
No
What are the two strands of DNA in human mito genome?
Light strand and heavy strand
What does human mito light strand encode?
1 protein and several tRNAs
What does human mito heavy strand encode?
12 proteins, ribosomal RNAs, and several tRNAs
where are tRNA genes placed on human mito heavy strand?
Between coding sequences so that they punctuate the genome
What does the human mito genome control region contain?
Two promoters and one origin of replication.
Where is human mito heavy strain transcribed from?
A “strong” promoter that produces many copies of the rRNAs
What does human mito weak promoter do?
Results in one long polycistronic transcript that is cleaved to liberate the tRNAs. After cleavage, transcripts are polyadenylated. In some cases, the first base of the polyA tail is the last base of the stop codon.
How does yeast mito genome compare to human mito DNA?
It has many more noncoding sequences. The tRNA genes in yeast mito are placed in clusters. The order of the genes is different than in mammalian mito. The genes have introns, some which encode transposases.
What two things get imported into mito from cytosol? What happens to them?
Fatty acids and pyruvate. They get oxidized.
What does oxidation of fatty acids and pyruvate generate in the Krebs cycle?
Acetyl CoA and NADH
What does NADH contribute to ETC?
Electrons
What does ETC do?
Pump protons into the inner membrane space, resulting in acidification of this space
How is ATP produced in mitochondria?
Protons move down their concentration gradient through ATP synthase and produce ATP.
What are the 3 major protein complexes of ETC?
NADH dehydrogenase complex, cytochrome c reductase, and cytochrome c oxidase
NADH dehydrogenase complex characteristics
22 protein subunits, 7 encoded by mitochondrial genes
What does NADH dehydrogenase complex pass electrons to?
The mobile carrier ubiquinone
What does carrier ubiquinone pass electrons to?
The cytochrome b-c1 complex
Characteristics of cytochrome b-c1 complex
15 protein subunits, 1 encoded by a mitochondrial gene
Where are electrons from cytochrome b-c1 complex passed to?
The mobile carrier cytochrome c
What does mobile carrier cytochrome c pass electrons to?
The cytochrome oxidase complex
Characteristics of cytchrome oxidase complex
8 protein subunits, 3 encoded by mitochondrial genes
what happens as electrons pass through the electron transport chain?
There are drops in free energy
How does ATP synthase produce ATP?
It uses the energy from the proton gradient to produce ATP
Characteristics of ATP synthase
12 subunits, two encoded by mitochondrial genes
Does the control region of the mammalian mito DNA maintain exact nucleotide sequences?
No
What does control region have?
One origin of replication and promoters for heavy strand and for light strand
What type of cells do mito defects often affect?
Those that have a large ATP requirement such as optic nerve and muscles
What diseases might be linked to mitochondrial defects?
Deafness, neuromuscular disease, alzheimers, parkinsons
What do some mito diseases result from?
Mutations in mito tRNA gene
Database of mitochondrial diseases
MITOMAP
When may mito diseases develop?
When somatic tissues acquire spontaneuous mutations in mito DNAs
Can mito repair their DNA?
They don’t have a strong DNA repair system
What do reactions on the cristae do to cause mutations?
Generate high levels of free radicals (superoxides)
What do mutations in the control region of human mito DNA do?
Suppress mito DNA replication and transcription
How many patients with mito disease have same mutation?
70-80%
How do mito mutations accumulate? What type of mutations are they?
With aging. They are somatic, not germline, mutations.
How do cells change as patients age?
They increase in heteroplasmy.
When do cells die because of mito disfunction?
Once a critical threshold is reached (loss of more than ~15% wild type mito
What is alzheimer’s disease?
Neurons die due to oxidative stress
What is amyloid beta-peptide (Abeta)?
A 40-42 amino acid peptide that is a component of amyloid plaques found in the brains of alzheimer’s patients.
How does Abeta interact with mitochondrial alcohol dehydrogenase?
It inhibits its function
How does alpha beta peptide enter mitochondria?
It is imported into mito and localizes to cristae.
What may cause mitochondrial diseases to develop in humans?
When somatic tissues acquire spontaneous mutations in mitochondrial DNAs. These mutations accumulate with aging.
What kind of gene is the mitochondrial DNA polymerase?
A nuclear gene
What two domains does the mitochondrial DNA polymerase have?
A polymerizing domain and a proofreading domain
What mito DNA polymerase domain was mutated in mice in an aging experiment?
The proof-reading domain
What happened to the mice that were homozygous for the mito DNA polymerase proof-reading domain mutation?
The mice aged much more rapidly than wild-type mice and lived only about half as long.
How many mitochondria does a cell have?
Many
How many copies of mito DNA does each mitochondrion have?
Many
Would a somatic mutation in one copy of the mito DNA have a detectable phenotype?
Not initially, but over time the mutant mito may come to dominate the population.
Until when is tissue normal when there are mito DNA mutations?
Until about 85% of mitochondrial DNA copies contain the mutation. Then the tissue crashes and function is lost.
Where are there numerous mutations found in mitochondria?
tRNA genes and protein coding genes
How many mitochondria do oocytes have?
~10,000
How are defective mitos treated in oocyte?
They are screened out and removed as the oocyte develops by a mechanism that is not understood.
What happens if defective mitos are not removed from oocyte?
Mutant mito DNA will pass to the progeny.
Is there a germline gene therapy for inherited mitochondrial diseases?
The 3-parent baby is a new approach that has been approved by british parliament and is being considered by FDA.
What is the 3-parent baby approach?
The chromosomes, contained on the meiosis I spindle, are removed from the oocyte that contains defective mitochondria. It is injected into a second oocyte (with normal mito) from which the spindle has been removed. The result is an oocyte with mito from one mother and nuclear genome from a second mother. The sperm can then fertilize the resulting egg.
What is heteroplasmy?
The condition in which some mitochondrial DNA copies in a cell are wild-type while other copies contain a mutation.
Two ways to prevent rare mitochondrial diseases?
Maternal spindle transfer; pronuclear transfer
Maternal spindle transfer
Chromosomes are removed from oocyte that has mutated mito DNA; these are added to an unfertilized donor egg that has had its nucleus removed; this fused egg is fertilized in vitro; the egg develops normally to form an embryo.
Pronuclear transfer
An egg with mutated mito DNA is fertilized in vitro; the resulting pronucleus is removed; this is transferred to a fertilized donor egg that has had its pronucleus removed; the fused egg develops normally.
Cytosolic S Ribosomes vs michondrial S ribosomes
80 S vs 55 S
Cytosolic rRNA vs mito rRNA
Both Lg rRNA, Sm rRNA
Cytosolic RNA vs mito RNA
5S RNA, 5.8S RNA vs nothing
cytosolic tRNAs vs mito tRNAs
60 tRNAs vs 22 tRNAs
inhibitors of mitochondrial ribosomes
chloramphenicol; erythromycin; vancomycin; lincomycin
chloramphenicol
broad-spectrum antibiotic causes aplastic anemia in children (non-reversible damage)
CIPRO
Antibacterial drug that can target mitochondria. CIPRO inhibits bacterial DNA gyrase and also inhibits mito DNA gyrase.
How are the very hydrophobic proteins encoded by mito DNA synthesized in the aqueous environment of the matrix and then imported?
Answer is in mito ribosome and its unusual structure. Proteins functionally replace rRNA to some extent. Ribosomal proteins dock the ribosome to the OXA complex. As they exit from the ribosome, nascent proteins are directed immediately into a channel in the PXA complex for insertion into the membrane.
What percent of mito proteins is encoded in nucleus?
More than 90%
Where are mRNAs translated?
On cytosolic ribosomes
When does transport into mitochondria occur?
Only post-translationally
What do proteins destined for import into mito contain?
A presequence (leader sequence, targeting sequence).
What happens to the mito protein presequence when the protein is folded?
The sequence becomes amphiphilic – plus charges on one side, nonpolar on the other.
How do mito protein leader sequences get to the TOM complex?
Receptors on the outer membrane bring these leader sequences to the TOM complex.
TOM complex
Translocase of the outer membrane; contains 7-8 proteins but only two proteins required form import: TOM 22, TOM 40
TIM complex
Translocase for the inner membrane – TIM 22, TIM 23
How do TOM and TIM work together?
They line up together in regions where the inner and outer membranes are closely spaced. An extension of the TIM complex interacts with the TOM complex.
What experiments revealed mitochondrial pre-sequence (targeting sequence)?
So basically, create a protein with radioactive amino acid labels. Add mito in early translation and late translation and see that it is smaller in SDS because it has been imported and pre sequence was cleaved off. If we treat the mito with protease to remove proteins from surface, or remove membrane potential, or deplete ATP, the mito can’t import the protein, so pre-sequence is never cleaved.
Cytosolic HSP70
Heat shock proteins and chaperones
Role of chaperones in cytosol
Bind mitochondrial precursor proteins (client proteins) in cytosol and keep them unfolded
What is required for HSP70 to release the client protein
ATP hydrolysis
What happens to N-terminus of protein when it is imported into mito?
It is threaded first through TOM complex and adjacent TIM complex. Then mito HSP70 in matrix binds the protein and pulls it into the matrix using ATP dependent “ratchet” mechanism.
What happens to protein once it has been imported into mito matrix?
Signal peptidase cleaves the signal peptide and the HSP70 continues to “pull” the remainder of the protein into the matrix.
What is required in mito matrix to fold proteins?
An HSP60 protein
What mechanisms are used to sort proteins into inner mito membrane?
Different mechanisms, including the OXA complex on the inner membrane. Some proteins enter matrix and then associate with the OXA complex to insert into the inner membrane.
How do proteins import into different mito compartments?
a. N-terminal signal sequence initiates import into matrix space (after going through TOM). A hydrophobic sequence binds to TIM 23 and stops translocation. Remainder of the protein is then pulled into the intermembrane space through TOM, and hydrophobic sequence is released into the inner membrane to anchor protein. B. inner membrane intergration – protein goes into matrix space completely. Signal sequence cleaved off, revealing hydrophobic sequence that uses OXA dependent pathway to insert itself in inner membrane and pull rest of protein out into intermembrane space. C. soluble intermembrane space proteins released into intermembrane space by a second signal peptidase which has active site in intermembrane space and removes hydrophobic signal sequence. D. soluble intermembrane proteins oxidized by Mia40 during import from outer membrane. Mia40 forms covalent intermediate through intermolecular disulfide bond, which pulls protein through TOM. Mia40 becomes reduced in process and is reoxidized by ETC, so it can catalyze next import round. E. multipass inner membrane proteins that function as metabolite transporters contain internal signal sequences and snake through TOM complex as a loop. Bind to chaperones in intermembrane space that guide them through TIM22.
What is TIM 22 specialized to do?
Insert multipass inner membrane proteins
How many pores does Tom complex have?
Two. They work together.
Which proteins can’t be removed from TOM complex for import?
TOM40 and TOM22
Role of peroxisomes
Like mito, play a role in oxidative pathway in cells
Do peroxisomes have DNA?
No
Do peroxisomes have a double membrane layer?
No. a single membrane.
How many enzymes do peroxisomes have? What do they do?
50-60 enzymes, most involved in detox reactions
two step peroxisome detox process
- substrates like uric acid and aa are oxidized to form H2O2. Very long-chain fatty acids are oxidized in detox reactions. 2. Catalase breaks down the H2O2.
How do fireflies use peroxisomal enzyme luciferase?
To oxidize a substrate luciferin to emit visible light from cells in the abdomen.
What causes zellweger syndrome?
Mutations in genes required for import of proteins into peroxisomes. Peroxisomes are not functional and patients die at an early age.
What happens to catalase antibody in some patients with zellweger’s?
Catalase antibody does not detect catalase in peroxisomes, but does detect PMP70 from the peroxisomal membrane. So peroxisome membranes are present but empty.
What was observed in some fusion combinations of fibroblasts from different patients?
Rescue or “complementation” of the defect.
How many genes have been identified that can cause zellweger’s when mutated?
18-20
How have genetic mutations that cause zellweger’s been used?
To identify the assembly steps for peroxisomes
What kinds of peroxisomes mutations are there?
Those that cause defects in 1 formation of peroxisomes 2. Import of proteins into peroxisomes and 3 function of protein (enzymes) in the peroxisomes.
Peroxisome biogenesis
Peroxisomal membranes are derived from vesicles budding from ER
How are additional proteins to peroxisomes?
By import from cytosol. Membrane and matrix proteins are imported.
How can peroxisomes fuse and divide?
By fission
What do peroxisomal matrix proteins contain?
A targeting sequence (PTS1) at the C-terminus which is recognized by Pex 5 receptor.
What receptor does Pex5 work with?
Pex14 receptor, located on peroxisome membrane.
What happens to matrix protein=Pex5 complex?
It is transferred to a set of membrane proteins (Pex 10, 12 and 2) that are necessary for translocation into the peroxisomal matrix by an unknown mechanism.
What happens to Pex5 after protein has been translocated?
It dissociates from the matrix protein and returns ot the cytosol, a process that involved the Pex2/10/12 complex and addl membrane and cytosolic proteins not shown.
Can folded proteins be imported into peroxisome?
Yes. Their targeting sequence is not removed in the matrix.
What genes encode peroxisomal proteins?
Nuclear genes
Where are peroxisomal proteins synthesized?
On cytosolic ribosomes and folded in cytosol.
What is SKL?
A targeting sequence at C terminus of peroxisomal proteins
What does Pex5 become during protein import?
Part of the translocation pore (transient pore)
Is unfolding of protein required for import into peroxisome?
No
How is large catalase tetramer imported into peroxisome?
In its folded state.
What, in general, do receptor proteins for peroxisome do?
They shuttle back and forth from the peroxisome to the cytosol to bind peroxisomal proteins and bring them to the peroxisome.
