Exam 2b Flashcards Preview

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Flashcards in Exam 2b Deck (279):
1

What do proteins define in each compartment of a cell?

function

2

where are localization signals in a protein

they are part of primary or secondary structure

3

nuclear localization signal (NLS)

directs proteins to nuclear pore complex (NPC)

4

what is in localization signal

proline followed by positive amino acids

5

where can localization sequence be

anywhere in protein

6

what size things can diffuse freely into nucleus

5kDa small molecules, dNTP, NTP, small proteins

7

what size things for sure need assistance getting into nucleus

30kDa, large proteins, RNAs

8

characteristics of nuclear porin

huge, has 30 proteins, many copies, approximately 120 Mda

9

what is NPC center like

gelatinous and disordered

10

how is NPC selective

for size

11

what is nuclear porin gate made of

glycine and phenylalanine, which are both nonpolar

12

key players in nuclear import/export

nuclear import receptor, nuclear export receptor, Ran, RanGTP, RanGDP, RanGAP, RanGEP

13

nuclear importer receptor

floating in cytosol, interacts with NLS, directs proteins to nuclear pore

14

Ran

monomeric G protein, binds receptor when bound to GTP in nucleus, shuttles import receptor back out

15

Ran-GAP

in cytosol, releases Ran from receptor GTP to GDP

16

Ran-GEF

in nucleus, converts Ran-GDP to Ran-GTP, maintains Ran-GTP

17

active and inactive form of Ran

Ran GTP is active/ Ran GDP is inactive

18

what limits NLS exposure

ligand binding/conformation

19

what do nuclear export receptors do

go into nucleus, pick up proteins with nuclear export signal and deliver it to cytosol

20

What role does Ran-GTP play in export

promotes cargo association

21

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

22

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.

23

endoplasmic reticulum

extensive network of membrane/ expansive and dynamic

24

smooth ER

lipid and steroid synthesis

25

rough ER

protein synthesis for entire endomembrane system

26

signal peptide (SP)

for entry into ER or for secretion of bacterial proteins

27

where is signal peptide and what is it?

at the N-terminus, a hydrophobic alpha helix, approximately 15-30 amino acids

28

when do most proteins enter ER

during translation, “co-translational”, reason for RER formation

29

what happens during ER signal sequence

translocation

30

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.

31

pH of lumen ER

Approximately 6

32

ph outside lumen of ER

7

33

2 types of ribosomes

free ribosomes and membrane bound ribosomes (coat rough ER)

34

Type I transmembrane protein

has signal peptide and is equal to or greater than 1 hydrophobic alpha helix

35

which side of start-transfer protein ends up facing cytosol?

the more positive side stays out

36

Type II transmembrane protein

No SP, has “internal start transfer” sequence

37

Post-translation

fully synthesized,then directed into ER

38

what translocator do bacteria, archea and eukaryotes all have

Sec 61

39

What complex do eukaryotes use for translocation post-translation

Sec 62, 63, 71, 72 complex

40

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.

41

How does BIP work

BIP is ATP driven. Release pull protein into lumen

42

sec Y

translocator complex used by bacteria

43

what feeds sec Y

Sec A ATPase with conformation changes that cause piston like motion in Sec A. only in bacteria.

44

two post translational modifications in ER

N-linked glycosylation; Disulfide bond formation

45

when does glycosylation happen? To what?

-as proteins enter or after;-both soluble and transmembrane proteins

46

sequence for attaching oligosaccharide

Asn-X-Ser/thr

47

how does N-glycosylation end

in three sugar residues

48

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

49

how does it prevent proteolysis

protein can have many oligosaccharides attached to it, and they shield it from proteases

50

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

51

what happens if protein never folds correctly

;chaperones, disulfide isomerases and lectins are involved
-go to cytosol;ubiquitylated, deglycosylated and degraded in proteasome

52

how does n-glycosylation work

Oligosaccharide is attached to PM, neighboring a growing peptide chain. Oligosaccharide protein transferase transfers oligosaccharide to peptide chain.

53

what forms SS bond

protein disulfide isomerase or pdi;-helps correct disulfide bonds to form

54

There are no disulfides in cytosolic proteins. Why?

because of reducing environment, which means there is glutathione that breaks SS bonds

55

where do SS bonds exist

only inside organelles and extracellularly

56

what happens to SS bonds in cytosol

become SH sulfhydryl groups

57

what do sulfhydryl groups mean to ER

a signe of incomplete disulfide bond formation

58

can there be SS bonds in nucleus

no. it has a reducing environment like cytosol

59

What is the endomembrane system?

ER, Golgi (has cisternae), PM, endosomes, lysosomes/vacuoles, vesicles, peroxisomes

60

what is entry point to endomenbrane system

ER

61

what make EM a system

exchange of materials

62

what kind of transportation does EM rely on

vesicular transport

63

what is pH of ER

6

64

is lumen of EM acidic or basic

acidic

65

what is pH of lysosome. Why?

5. due to H+ATPase, which adds protons

66

what happens to pH as proteins move farther into EM

gets more acidic

67

three parts of vesicular transport

formation (budding), movement to target membranes, fusion with target membranes

68

what are players in vesicle formation

cargo, receptor, adaptor, coat proteins, dynamin

69

vesicle formation steps

initiate bud, coat proteins drive vesicle formation, adaptors have clathrin triskeleon, then dynamin pinches off vesicle, coat falls off

70

what does dynamin use to pinch off vesicle

GTP; wraps around and pinches off

71

are adaptors always open

no, they are locked sometimes

72

with what do different adaptors associate

different membranes and different coat proteins via phosphoinositides in cytosolic leaflet

73

what can be modified in phosphoinositides

three OH groups after carbon 1

74

how are different PIPs produced

phosphorylation of 1, 2 or 3 carbons can form a variety of species

75

do animal cells only have one PI or PIP

no, they have various

76

what catalyzes PIP production

PIP phosphatase

77

where do PIPs live

in different membranes and different domains

78

what are PIPs associated with

specific vesicle transport events

79

vescicle membrane steps

Has PIP, fuses with PM, PI kinase adds P, recruits adaptor protein, initiate clathrin coated pit, vesicle hydrolizes and loses coat

80

how do coat proteins drive vesicle formation

via autoassembly

81

what is triskeleon made of

three heave clathrin chains and three light chains

82

types of coat proteins

clathrin, COPI, COPII

83

what do different coat proteins do

select different cargo

84

how does vesicle move

via cytoskeleton, does not float, motor proteins move vesicle along microtubule towards, for example, cis golgi

85

what does vesicle fusion with target membrane require

Rab and SNAREs

86

what form of Rab is active

Rab-GTP

87

what does Rab-GTP do

identifies target membrane

88

what do SNAREs do

drive vesicle fusion

89

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

90

Coiled coils

nonpolar wrap around each other (two alpha helices, eg.);can join both in parallel and antiparallel direction

91

Types of SNAREs

v-SNARE –vesicular-attaches to vesicle;t-SNARE – target membrane – attaches to membrane

92

Vesicle fusion Steps

1. 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)

93

what happens to membrane orientation during fusion

;Membrane orientation is maintained with each budding and fusion event;one leaflet always faces cytosol

94

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.

95

dissociation of SNARE pairs by NSF after fusion

;NSF binds to SNARE complex;has accessory proteins help; hydrolyzes ATP to pry SNAREs apart

96

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

97

charge of amino acid terminus

positive

98

golgi apparatus

– condensed stacks of membrane near cell center

99

golgi parts

cis golgi – receives from ER; trans golgi – stuff leaves golgi from here

100

major functions of golgi

1. modification/synthesis of glygolipids and glycoproteins;2. golgi is post office

101

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

102

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

103

Two types of secretion:

. constitutive and regulated

104

constitutive secretion

-all cells do constitutive exocytosis to maintain PM and extracellular space

105

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

106

Exocytosis vs endocytosis in endomembrane system:

In growing, dividing cells : exo > endo;In nongrowing cells : exo = endo

107

Endomembrane system is:

1. 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)

108

Zip code for secreted proteins:

N-| SP | ------------------------------ |-C; SP and nothing else – protein will be secreted from cell

109

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)

110

Trip to lysosome

ER Golgi endosome lysosome

111

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

112

List of acid hydrolases:

Nucleases Proteases Glycosidases Lipases Phosphatases Sulfatases Phospholipases

113

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

114

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

115

Pinocytosis

-small stuff-means cell drinking-via vesicular trafficking-all eukaryotes-regulated vs constitutive

116

constitutive pinocytosis reason

to rejuvenate PM and extracellular space

117

regulated pinocytosis reason

for specific material, receptor mediated

118

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

119

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

120

homotypic fusion

Endosomal fusion

121

where do digestive enzymes fuse with endosomes

TGN

122

what do endosomes have internally

multivesicular; have RNA

123

Phagocytosis =

-large stuff-cell eating-foreign cells, cell debris (digested in lysosome)

124

what does phagocytosis

white blood cells, amoebae, protists

125

Autophagy:

self eating-for damaged organelles or starvation conditions, especially mitochondria

126

Four degradation pathways

1. 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)

127

Inner mito membrane characteristics

-has no cholesterol, has infolding to form cristae, has electron transport system and ATP synthase, has enzymes for lipid synthesis

128

why is inner membrane space more acidic than matrix space?

Protons are pumped into this space by the complexes in the ETC.

129

Outer membrane characteristics

-has cholesterol, contains import receptors and porins for import of large molecules

130

What encloses matrix compartment?

Inner membrane

131

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.

132

What is inner membrane compartmentalized into? 2

Inner boundary membrane and crista membrane.

133

Three distinct spaces in the mitochondrium

Inner membrane space, crista space, and the matrix.

134

What is crista junction?

Joins cristae to inner boundary membrane

135

What led to an aerobic eukaryotic cell?

The uptake of a bacterial cell with ability for respiration

136

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.

137

To where did genes from endosymbiont move? How may proteins do modern mitochondrial genomes code for?

To the nucleus. 13 proteins.

138

What is Rickettsia prowazekii?

An obligate intracellular parasitic bacterium that causes typhus.

139

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.

140

What gave rise to chloroplasts?

An endosymbiotic oxygen loving cyanobacterium (cyan)

141

From what did mitochondria arise?

Alpha-proteobacterium

142

What are the nearest relatives of mitochondria?

Three closely related groups of alpha-proteobacteria – the rhizobacteria, agrobacteria, and rickettsias.

143

What kind of photosynthesis did ancestral fermenting bacteria do?

H2S

144

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.

145

What happened before the rise of cyanobacteria?

H2O photosynthesis

146

What three types of bacteria did purple nonsulfur bacteria break into?

Beta proteobacteria, alpha proteobacteria, and gamma proteobacteria

147

What is extranuclear inheritance?

Phenotype does not segregate with nuclear genes.

148

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.

149

How many mitochondria do cells contain?

Multiple

150

How many copies of DNA does each mitochondrion contain?

Multiple

151

What did yeast progeny cells contain mitochondria wise?

They were heteroplasmic – contained multiple mitochondria, some mutant and some wild type.

152

When did mutant yeast phenotype get expressed?

When WT mito fell into minority.

153

What was the name of the mutant Neurospora crassa fungus?

Poky

154

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.

155

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.

156

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.

157

What DNA do human mitochondria contain?

A circle 5 microns in length, containing 16 kb of DNA.

158

How is yeast mito DNA different from human?

It is five times larger, but has the same amount of genetic information.

159

What do mitochondrial genomes encode?

All the tRNAs and ribosomal RNAs necessary for translation in the mitochondria.

160

How many tRNA genes are there in mammalian mito?

22 tRNA genes

161

How long is human mito DNA? Yeast mito DNA?

5microM vs 25 microM

162

Are there introns in human mito DNA?

No

163

What are the two strands of DNA in human mito genome?

Light strand and heavy strand

164

What does human mito light strand encode?

1 protein and several tRNAs

165

What does human mito heavy strand encode?

12 proteins, ribosomal RNAs, and several tRNAs

166

where are tRNA genes placed on human mito heavy strand?

Between coding sequences so that they punctuate the genome

167

What does the human mito genome control region contain?

Two promoters and one origin of replication.

168

Where is human mito heavy strain transcribed from?

A “strong” promoter that produces many copies of the rRNAs

169

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.

170

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.

171

What two things get imported into mito from cytosol? What happens to them?

Fatty acids and pyruvate. They get oxidized.

172

What does oxidation of fatty acids and pyruvate generate in the Krebs cycle?

Acetyl CoA and NADH

173

What does NADH contribute to ETC?

Electrons

174

What does ETC do?

Pump protons into the inner membrane space, resulting in acidification of this space

175

How is ATP produced in mitochondria?

Protons move down their concentration gradient through ATP synthase and produce ATP.

176

What are the 3 major protein complexes of ETC?

NADH dehydrogenase complex, cytochrome c reductase, and cytochrome c oxidase

177

NADH dehydrogenase complex characteristics

22 protein subunits, 7 encoded by mitochondrial genes

178

What does NADH dehydrogenase complex pass electrons to?

The mobile carrier ubiquinone

179

What does carrier ubiquinone pass electrons to?

The cytochrome b-c1 complex

180

Characteristics of cytochrome b-c1 complex

15 protein subunits, 1 encoded by a mitochondrial gene

181

Where are electrons from cytochrome b-c1 complex passed to?

The mobile carrier cytochrome c

182

What does mobile carrier cytochrome c pass electrons to?

The cytochrome oxidase complex

183

Characteristics of cytchrome oxidase complex

8 protein subunits, 3 encoded by mitochondrial genes

184

what happens as electrons pass through the electron transport chain?

There are drops in free energy

185

How does ATP synthase produce ATP?

It uses the energy from the proton gradient to produce ATP

186

Characteristics of ATP synthase

12 subunits, two encoded by mitochondrial genes

187

Does the control region of the mammalian mito DNA maintain exact nucleotide sequences?

No

188

What does control region have?

One origin of replication and promoters for heavy strand and for light strand

189

What type of cells do mito defects often affect?

Those that have a large ATP requirement such as optic nerve and muscles

190

What diseases might be linked to mitochondrial defects?

Deafness, neuromuscular disease, alzheimers, parkinsons

191

What do some mito diseases result from?

Mutations in mito tRNA gene

192

Database of mitochondrial diseases

MITOMAP

193

When may mito diseases develop?

When somatic tissues acquire spontaneuous mutations in mito DNAs

194

Can mito repair their DNA?

They don’t have a strong DNA repair system

195

What do reactions on the cristae do to cause mutations?

Generate high levels of free radicals (superoxides)

196

What do mutations in the control region of human mito DNA do?

Suppress mito DNA replication and transcription

197

How many patients with mito disease have same mutation?

70-80%

198

How do mito mutations accumulate? What type of mutations are they?

With aging. They are somatic, not germline, mutations.

199

How do cells change as patients age?

They increase in heteroplasmy.

200

When do cells die because of mito disfunction?

Once a critical threshold is reached (loss of more than ~15% wild type mito

201

What is alzheimer’s disease?

Neurons die due to oxidative stress

202

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.

203

How does Abeta interact with mitochondrial alcohol dehydrogenase?

It inhibits its function

204

How does alpha beta peptide enter mitochondria?

It is imported into mito and localizes to cristae.

205

What may cause mitochondrial diseases to develop in humans?

When somatic tissues acquire spontaneous mutations in mitochondrial DNAs. These mutations accumulate with aging.

206

What kind of gene is the mitochondrial DNA polymerase?

A nuclear gene

207

What two domains does the mitochondrial DNA polymerase have?

A polymerizing domain and a proofreading domain

208

What mito DNA polymerase domain was mutated in mice in an aging experiment?

The proof-reading domain

209

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.

210

How many mitochondria does a cell have?

Many

211

How many copies of mito DNA does each mitochondrion have?

Many

212

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.

213

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.

214

Where are there numerous mutations found in mitochondria?

tRNA genes and protein coding genes

215

How many mitochondria do oocytes have?

~10,000

216

How are defective mitos treated in oocyte?

They are screened out and removed as the oocyte develops by a mechanism that is not understood.

217

What happens if defective mitos are not removed from oocyte?

Mutant mito DNA will pass to the progeny.

218

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.

219

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.

220

What is heteroplasmy?

The condition in which some mitochondrial DNA copies in a cell are wild-type while other copies contain a mutation.

221

Two ways to prevent rare mitochondrial diseases?

Maternal spindle transfer; pronuclear transfer

222

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.

223

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.

224

Cytosolic S Ribosomes vs michondrial S ribosomes

80 S vs 55 S

225

Cytosolic rRNA vs mito rRNA

Both Lg rRNA, Sm rRNA

226

Cytosolic RNA vs mito RNA

5S RNA, 5.8S RNA vs nothing

227

cytosolic tRNAs vs mito tRNAs

60 tRNAs vs 22 tRNAs

228

inhibitors of mitochondrial ribosomes

chloramphenicol; erythromycin; vancomycin; lincomycin

229

chloramphenicol

broad-spectrum antibiotic causes aplastic anemia in children (non-reversible damage)

230

CIPRO

Antibacterial drug that can target mitochondria. CIPRO inhibits bacterial DNA gyrase and also inhibits mito DNA gyrase.

231

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.

232

What percent of mito proteins is encoded in nucleus?

More than 90%

233

Where are mRNAs translated?

On cytosolic ribosomes

234

When does transport into mitochondria occur?

Only post-translationally

235

What do proteins destined for import into mito contain?

A presequence (leader sequence, targeting sequence).

236

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.

237

How do mito protein leader sequences get to the TOM complex?

Receptors on the outer membrane bring these leader sequences to the TOM complex.

238

TOM complex

Translocase of the outer membrane; contains 7-8 proteins but only two proteins required form import: TOM 22, TOM 40

239

TIM complex

Translocase for the inner membrane – TIM 22, TIM 23

240

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.

241

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.

242

Cytosolic HSP70

Heat shock proteins and chaperones

243

Role of chaperones in cytosol

Bind mitochondrial precursor proteins (client proteins) in cytosol and keep them unfolded

244

What is required for HSP70 to release the client protein

ATP hydrolysis

245

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.

246

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.

247

What is required in mito matrix to fold proteins?

An HSP60 protein

248

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.

249

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.

250

What is TIM 22 specialized to do?

Insert multipass inner membrane proteins

251

How many pores does Tom complex have?

Two. They work together.

252

Which proteins can’t be removed from TOM complex for import?

TOM40 and TOM22

253

Role of peroxisomes

Like mito, play a role in oxidative pathway in cells

254

Do peroxisomes have DNA?

No

255

Do peroxisomes have a double membrane layer?

No. a single membrane.

256

How many enzymes do peroxisomes have? What do they do?

50-60 enzymes, most involved in detox reactions

257

two step peroxisome detox process

1. 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.

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How do fireflies use peroxisomal enzyme luciferase?

To oxidize a substrate luciferin to emit visible light from cells in the abdomen.

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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.

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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.

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What was observed in some fusion combinations of fibroblasts from different patients?

Rescue or “complementation” of the defect.

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How many genes have been identified that can cause zellweger’s when mutated?

18-20

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How have genetic mutations that cause zellweger’s been used?

To identify the assembly steps for peroxisomes

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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.

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Peroxisome biogenesis

Peroxisomal membranes are derived from vesicles budding from ER

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How are additional proteins to peroxisomes?

By import from cytosol. Membrane and matrix proteins are imported.

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How can peroxisomes fuse and divide?

By fission

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What do peroxisomal matrix proteins contain?

A targeting sequence (PTS1) at the C-terminus which is recognized by Pex 5 receptor.

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What receptor does Pex5 work with?

Pex14 receptor, located on peroxisome membrane.

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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.

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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.

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Can folded proteins be imported into peroxisome?

Yes. Their targeting sequence is not removed in the matrix.

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What genes encode peroxisomal proteins?

Nuclear genes

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Where are peroxisomal proteins synthesized?

On cytosolic ribosomes and folded in cytosol.

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What is SKL?

A targeting sequence at C terminus of peroxisomal proteins

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What does Pex5 become during protein import?

Part of the translocation pore (transient pore)

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Is unfolding of protein required for import into peroxisome?

No

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How is large catalase tetramer imported into peroxisome?

In its folded state.

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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.