Bio 314 - Exam 2

mitochondria

translocator

Mediates protein mov’t across membrane (outer: TOM, inner: TIM)

precursor protein

Protein prevented from being folded due to chaperones, has not yet entered the organelle

Steps to mitochondria protein translocation

1. precursor interacts with TOM import receptor, ATP removes cytosolic hsp chaperones

2. Protein passes through outer membrane, signal sequence interacts w/TIM23 on inner membrane

3. Mitochondrial hsp70 uses ATP to “ratchet” protein into matrix

4. hsp 70 helps protein fold using ATP hydrolysis

Mitochondria single pass inner membrane import

Only N-terminus enters into matrix before hydrophobic stop-transfer sequence prevents further entry. Signal sequence is cleaved and remains in inner-membrane space

Mitochondria inter-membrane import

After becoming a single pass inner-membrane protein, protease cleaves membrane anchor releasing protein into inter-membrane space

Mitochondria multi-pass intermembrane import

1. Protein internal sequence enters TOM as a loop

2. Intermembrane chaperones guide protein to TIM22

3. TIM22 inserts protein into inner membrane

Mitochondria outer-membrane import

1. Protein fully enters intermembrane space via TOM

2. Chaperones bind to prevent folding

3. SAM complex inserts protein into outer membrane

4. Porin forms (pore shaped beta-barrel)

chloroplast

What do mitochondria/chloroplast transposition have in common?

Require energy, occur after translation, protein have signal sequences, distinct translocators

How is chloroplast transposition different from mitochondria?

Have TOC/TIC rather than TOM/TIM, extra membrane, protein with extra signal sequence to enter thy. membrane, different energy sources (H+ gradient, ATP, GTP)

Endoplasmic reticulum transposition

Ribosomes attached to rough ER allow co-translational transport. All transmembrane proteins and lipids form here

signal recognition particle (SRP)

Binds to mRNA as it is being translated and changes conformation to curl around the ribosome and pause translation

How are proteins co-translationally transported to the ER?

1. Translation begins in cytosol, ER signal sequence is created

2. SRP binds to ER signal sequence to pause translation

3. Gets ferried to ER and attaches to SRP receptor on ER membrane

4. SRP is removed, translation resumes

single pass ER membrane proteins

1. Transmembrane segment of protein directs insertion (of C or N terminus) into ER membrane

2. Sec61-SRP receptor mediates single-pass of protein

3. ER signal sequence and transmembrane segment creates complex domains on either side of the membrane

multipass ER membrane protein

1. Internal signal sequence starts translocation until encountering hydrophobic stop

2. Alternating stop/start sequence locks segment into bilayer

exocytosis

Secretory pathway, “excretes” proteins, carbs, lipids to PM/extracellular space

endocytosis

Intake of nutrients/transfer from one organelle to another through endosomes, vesicles form by budding

secretory pathway

Cargo moved out from inside the cell. Starts with ER, goes to golgi, then the ECF

endocytic pathway

Cargo moves in from the ECF. Starts at the PM → lysosome

retrieval pathway

Cargo is moved back and fourth to create balance, molecules are recycled or returned to compartment of origin. Relies on ER retrieval signal to package COPI coated vesicles

coated vesicle

Have a distinct protein cage that’s discarded before reaching target. Inner coat concentrates proteins for transport, outer coat shapes vesicle with curved lattice work

clathrin

Protein that transfers vesicle from golgi→PM. Clathrin subunits form triskelion, adaptor proteins of inner layer bind coat and transmembrane proteins

Vesicle coat formation

1. Cargo binds TM and adapter proteins

2. Coat recruitment GTPase in cytosol is GDP-bound (inactive)

3. Membrane embedded GEF exchanges GDP→GTP

4. GTP recruits adaptor coat proteins to initiate budding

coat-recruitment GTPases

ARF, Rab7, and SarI proteins that bind to GDP (inactive, turns to GAP) or GTP (active, turns to GEF) and ensure coat proteins assemble where/when needed

GAP (inactive switch)

The result of GDP nucleotide binding to the coat-recruitment GTPase, found in ER lumen

GEF (active switch)

The result of GTP nucleotide binding to the coat-recruitment GTPase, found in the cytosol

Rab proteins

On vesicle coat, directs to specific spots on target membrane by anchoring and binding to Rab effectors. Rab-GTP when attached to vesicle, Rab-GDP when recycled through cytosol

Rab effectors

On target membrane, tethers to Rab-GTP protein on vesicle

trans SNARE complex

V-snares on vesicle membrane and T-snares on target wrap together and release energy in a favorable union

NSF proteins

Use ATP hydrolysis to unwind trans-SNARE complex

Function of COPI coated vesicles

Retrieval pathway, return to ER from golgi containing proteins that have escaped their organelle, cargo receptors, and v-snares

vesicular tubular clusters

Several vesicles leaving the ER membrane fuse together on their way to the golgi

Cisternal maturation model

When the vesicular tubular cluster approaches the cis-golgi network, it forms a premature cisternae and the layers of cisternae shift until the trans-golgi network buds off new vesicles

vesicle transport model

As the vesicular tubular cluster approaches the cis-golgi network, the vesicles fuse and reform throughout all of the separate fixed cisternae

How does the golgi sort lysosomal proteins for transport?

Proteins/lipids are glycosylated as they leave the ER, hydrolases have unique marker (M6P) added to their oligosaccharide, clathrin adaptor proteins seek marker to load cargo into vesicle

autophagy

Cell “eats” part of itself to dispose unneeded materials. Selective=eating specific cargo or nonselective=eating bulk cytoplasm as a survival mechanism

1. Trigger from a signaling molecule

2. Formation of vesicle and extension to create autophagosome

3. Closure of lipid bilayer around autophagosome

4. Fusion w/lysosome

receptor mediated endocytosis

Internalization of something extracellular, occurs when extracellular cargo binds to transmembrane receptors that enter the cell through clathrin coated vesicles

phagocytosis

Vesicles (phagosomes) feed on extracellular dead cells/destroy foreign invaders

1. Triggered by specific cargo

2. Diameter of the phagosome adjusts to size of particle

3. Fuses w/lysosome

Why is ATP a main form of energy?

ATP is an energy coupling agent that creates an exergonic process to drive an endergonic one, energy is stored in bonds b/t Pi

cellular respiration

Oxidizes glucose into CO2 and H+ that will create a proton gradient and ~30 ATP

glycolysis (step 1 cellular respiration)

Splits 1 glucose into 2 pyruvate molecules, 2 ATP, and 2 NADH

pyruvate oxidation & citric acid cycle (step 2 cellular resp.)

2 pyruvate is transferred to mitochondria, pyruvate dehydrogenase converts it to 2 acetyl CoA and 2 NADH

Citric acid cycle produces 6 NADH, 2 FADH2, and 2 ATP from 2 acetyl CoA

electron transport chain & oxidative phosphorylation (step 3 cellular resp.)

Pass on FADH2 and NADH electrons to activate four e- driven proton pumps. Each NADH moves 10 H+ and FADH2 moves 6 H+

During oxidative phosphorylation ATP Synthase makes ATP using the H+ gradient.

How do electrons generate ATP in mitochondria?

H+ gradient across mito membrane creates (+) charge in cristae and (-) charge in matrix, starts a chemiosmotic process where energy stored in gradient drives ATP synthase

Order of electron transport H+ pumps

NADH dehydrogenase→ succinate dehydrogenase →cytochrome C reductase→ cytochrome C oxidase

NADH dehydrogenase (ETC)

L-shaped complex w/hydrophobic arm embedded in mito. membrane containing proton pumps, and hydrophilic arm in matrix that transfers the e- from NADH to ubiquinone. NADH oxidizes to NAD+

succinate dehydrogenase (ETC)

Enzyme that generates FADH2 inside matrix, FADH2 transfers e- to ubiquinone and oxidizes to FAD

Q-cycle (ETC)

Ubiquinone shuttles 2 e- from the NADH dehydrogenase complex and FADH2 to cytochrome C, and in turn accepts 2H+ to become ubiquinol. One e- goes to ubiquinol while the other is recycled to the intermembrane space

cytochrome C reductase (ETC)

Houses the Q-cycle, and for every electron shuttled by ubiquinone 2H+ are pumped into cristae which creating high redox energy to be stored in an electrochemical gradient

cytochrome C oxidase (ETC)

Last location for cytochrome C where e- is transferred to bimetallic center (holds e- until O2 can safely be oxidized to H2O)

Mechanism of ATP synthase

H+ moves down electrochemical gradient to spin rotor, which generates 3 ATP per turn. Located at sinus of cristae where H+ are attracted to creating a proton sink

ATP synthase

light reaction

uses light to split H2O and create O2, NADPH, and ATP

Calvin cycle

uses CO2, NADPH, and ATP to create sugar and H2O

What are the primary e- acceptors in PSII and PSI?

plastoquinone and ferredoxin

What is the path of e- through the chloroplast?

antennae compex→PSII (plastoquinone)→cytochrome complex (ETC)→plastocyanin→ferredoxin→ferredoxin NADP+ reductase

PSII (step 1 of photo.)

Photons excite e- in P680. PSII splits H20, passes e- to plastoquinone

cytochrome complex/ETC (step 2 of photo.)

Receives e- from plastoquinone, uses it to pump H+ into thylakoid, passes e- to plastocyanin

PSI (step 3 of photo.)

Plastocyanin passes e- to P700 in PSI, returning it to its normal state

What happens when photons are captured by the light harvesting complex?

Both PSII and PSI pigment molecules release an e- that is passed to the primary electron acceptors

ferredoxin NADP+ reductase (step 4 of photo.)

Ferredoxin gives e- to ferredoxin NADP+ reductase so it can generate NADPH and ATP

C-fixation (Step 1 Calvin cycle)

CO2 and RuBP are catalyzed by RuBisCo to create 3-PGA

Reduction (Step 2 Calvin cycle)

ATP and NADPH from photosynthesis convert 3-PGA into G3P (= 1 glucose that can continue on the the citric acid cycle)

Regeneration (Step 3 Calvin cycle)

Resupplies the cycle with more RuBP. 3 ATP convert remaining G3P molecules back to RuBP

Function of actin

Maintain cell shape, function in whole cell locomotion by projecting cell outwards towards a substrate or object (amoeba moving to engulf another organism), form contractile ring during cytokinesis.

Function of microtubules

hollow, stiff, strong, intracellular transport, motor proteins “walk” along, determine location of organelles, form mitotic/meiotic spindles that separate chromosomes during anaphase

Function of intermediate filaments

In specialized cases that require strength and flexibility, fingernails, hair, exoskeletons, skin capable of being stretched without breaking

actin

Direction of actin growth

F-actin grows point(-)→barb(+) fastest, G-actin carries ATP which spontaneously hydrolyzes to ADP and energy is stored inside filament

thymosin

Accessory protein that binds to actin monomer to prevent interaction b/t (+) and (-) ends

profilin

Competitively binds w/thymosin to enhance actin monomers to bind with (+) ends

ARP 2/3 complex (Nucleating proteins)

Mimics the (-) end of actin to promote rapid growth at (+) end, can attach to sides of filaments to create branching network of filaments

Formins (nucleating protein)

Associate with (+) end of actin proteins, create unbranched filaments and form thick parallel bundles of actin (cables)

(+) ends of actin reach z-disk, with (-) ends at the myosin line

Muscle sarcomere

Process of muscle contraction

Muscle uncontracted: Ca absent, tropomyosin on actin

1. Electrical signal opens voltage gated Ca channels, Ca+ released from sarcoplasmic reticulum

2. Ca+ removes tropomyosin complex, myosin binds actin

3. Conformation change creates power stroke, ADP & Pi released

4. ATP binds to myosin, myosin releases actin (+) end, ATP hydrolyzed

Cap2

accessory protein to prevent depolymerization of actin

Myosin

motor protein w/globular head, contains ATPase and generates force

monomers=tubulin proteins, (-) alpha end is rapid growth and dissociation of subunits

microtubule

alpha tubulin (-)

Attracts GTP cap which maintains straight, strong, lateral contacts. GTP=growing form

beta tubulin (+)

Attracts GTP or GDP, GDP creates curved unstable protofilaments that weaken microtubule bond through elastic strain

Dynamic instability (microtubules)

rapid conversion from growing to shrinking, occurs randomly or controlled by proteins

catastrophe (microtubules)

loss of GTP cap

rescue

regaining of GTP cap

Microtubule formation

Rate of growth depends on rate of GDP hydrolysis and rate of tubulin-subunit addition

(+) beta proteins join with (-) alpha proteins

nucleation

Rate limiting step in forming actin where subunits must form a stable nucleus of monomers to begin a new filament

microtubule organizing centers (MTOC)

Intracellular location near nucleus of the cell where microtubule nucleation occurs at the (-) alpha end. Gamma-tubulin ring complex anchors and caps slow growing strand to assist in growth at the (-) end

kinesins

Move toward (+) end of microtubule:

1. leading head exchanges ADP for ATP, anchors to microtubule

2. lagging head hydrolyzes ATP, releases Pi and microtubule

3. New ATP binding creates conformation change in neck linker to step

dynein

Move toward (-) end of microtubule:

1. ATP binds, head releases microtubule and conformation change in neck linker pushes head forward

2. ATP hydrolyzes, leading head binds to microtubule

3. Pi release returns dynein to original conformation to create power stroke

axonemal dynein

Function in beating of cilia and flagella

cytoplasmic dynein

Function in organelle and vesicle trafficking, centrosome positioning during cellular movements

intermediate filament

Structure of intermediate filaments

Central rods with NH2 and COOH ends twist around each other, do not have polarity (no + or - end) and cannot bind to motor proteins. Easy to bend, difficult to break apart

3 steps for cellular locomotion

1. Protrusion→Actin formation forces PM outward

2. Attachment→Actin connects PM to substrate

3. Traction→Actin & myosin contract cell forward, microtubules organize/propel internal cell contents forward, intermediate filaments make cell strong, stable, flexible