Cell bio exam 2

- protein translocation, NRG conversion, membrane transport & cytoskeleton

mechanisms for transport (3)

gated, protein translocation, vesicular transport

gated transport

in and out nucleus, bidirectional

protein translocation

across and into membrane, unidirectional

vesicular transport

transport vesicles, some bidirectional

the ER!

• huge (1/2 membrane)

• divided into smooth and rough regions\

• expansive

smooth ER

narrow tubules

lipid formation

drug detox

rough ER

elongated sheets with ribosomes

protein processing

studying ER using density centrifugation

allows for fractionalization

rough ER separates from smooth components

rough ER at bottom of tube

led to signal hypothesis

signal hypothesis

main idea: secreted vs cytosolic proteins

scientists put mRNA and free ribosomes together. free ribosomes from cytosol resulted in a match for predicted size of the protein. free ribosomes secreted did not match and were slightly bigger because they retain their signal sequence (not clipped by peptidase)

co translational protein translocation

making protein at same time of moving into target

translocator proteins used!

post-translational protein translocation

ribosomes generate protein in cytosol

chaperone proteins used!! keeps primary structure intact to bring to ER membrane

2 populations of ribosomes

1. membrane bound

2. free ribosomes

both create flexibility. cell can divert pool to fit demands of cell.

membrane bound ribosomes

• dock on ER membrane, eventually fall off and go back to pool

• less of these

• co-translational

• can dock and anchor

free ribosomes

• translation in cytosol

protein translocation steps

1. ribosome binds to SRP

2. ribosome gets mRNA segment

3. translation starts and produces small ER SS

4. translation stops

5. SRP binds to SS, goes to ER, docks to receptor

6. receptors recruit translocators

7. translocator opens and translocates protein across

8. SRP released from receptor and recycled through new cycle

if in cytosol: chaperones help pull and prevent misfolding!

SRP

protein w specific binding pocket for SS

Sec61

most common protein translocator

translocation with transmembrane segments

• stops translocation, signals translocator to release protein

• segment embedded in membrane

• directionality based on charges

• ONLY single pass orients so negative charge is inside membrane (in lumen)

multipass transmembrane proteins

• SS is not on N terminus

• INTERNAL SS. SS is not cleaved off

• transmembrane segment acts as hydrophobic stop sequence, triggers translocator release

• numerous internal start/stop sequences make it multi pass

post-translational translocation (cytoplasm protein where SS hidden inside and not directed to ER)

example: SNARE proteins/tail anchored proteins

SNARE proteins / tail anchored

• C-terminus embedded in membrane

• pre-targeting complex recognizes tail, binds

• recognized by chaperone Get3

• Get3 takes to ER membrane

Get3

common chaperone protein used in posttranslational protein translocation (in cytosol, no SS)

misfolded ER proteins

1. N-linked polysaccharides

2. polyubiquitin chains

N-linked polysaccharides

sugar chains that attach to proteins that check if protein is folding correctly

protein goes through glycosylation and glucose trimming

polyubiquitin chains

if reprocessed and protein still not folded, chaperones move it out of ER, and tag with polyubiquitin

recognized and sent to proteosome where it is broken down and turned into polypeptides and AA to be recycled

mitochondria (general)

structurally similar to gram negative bacteria (double membrane)

• inner membrane highly folded with cristae (intermembrane space)

• matrix

chloroplasts (general)

stroma - matrix like space

thylakoids - stacked membrane bound structures. a target for proteins

mitochondria translocation requirements

1. signal sequence

2. protein translocators specific to mitochondria

mitochondria different protein SS

• matrix space - SS on N-terminal

• outer/inner membrane - internal SS

protein translocators in different areas of mitochondria

• outermembrane: TOM, SAM

• inner membrane: TIM22, TIM23, OXA

post translational translocation needs (mitochondria)

1. chaperones to prevent misfolding and help bring to target

2. NRG. hydrolysis of ATP allows protein to lose chaperones and travel through TOM—>TIM23—>Matrix—>signal peptidases clip SS and protein folds

different forms of energy to move proteins (4)

1. ATP hydrolysis

2. electrochemical gradient

3. ATP hydrolysis (second time)

4. redox potential

ATP hydrolysis as NRG source to move proteins

1. first time it removes chaperones

2. second time: the same chaperones can use it to change conformation and get through the inner membrane (ex. mitochondrial hsp70)

mitochondrial hsp70

uses ATP hydrolysis so chaperones can change conformation and get through inner membrane

electrochemical gradient as NRG source for moving proteins

• positive in inner membrane (H+ flux after making ATP)

• negative inside matrix

• more positively charged amino acid side chains on proteins are attracted to the matrix

redox potential as NRG source of moving proteins

mia40—>intermembrane space

mia40 protein

• looks for sulfur side chains, oxidizes them to create disulfide bonds

• helps stabilize tertiary structure

• proteins for intermembrane space

• mia40 reduces itself in process

anchoring in inner mitochondrial membrane scenarios (4)

1. single N-terminal SS

2. 2 sequential N-terminal SS

3. post translational proteins from mitochondria

4. post translational proteins from cytosol

single N-terminal SS - docking in inner mitochondrial membrane

• directly followed by transmembrane segment

• stops translocation in TIM23

• releases protein into surrounding membrane

• inner: hydrophobic segment

2 sequential N-terminal SS - docking in inner mitochondrial membrane

• second one doesnt stop translocation

• goes all the way into matrix

• peptidase clips off SS, exposing the second

• chaperones take protein to another translocator in OXA

• remaining portion in inner membrane space

post translational proteins - docking in inner mitochondrial membrane

• made in matrix

• uses OXA complex to anchor into inner membrane

• efficient

multi-pass transmembrane proteins - inner membrane of mitochondria

• internal SS —> heavily embedded

• requires chaperones to direct to TIM22 complex which stitches proteins into membrane

inserting proteins in outer membrane of mitochondria

• similar bacterial mechanisms

• beta barrels

• porins

• SAM translocator

• intermembrane space

beta barrels

porous

common for inserting proteins in outer membrane of mitochondria

porins

in outer membrane of mitochondria to allow for free movement of molecules

less selective

bacterial mechanisms for outer membrane

• BAM translocator

• proteins start in cytosol

• still use chaperones

• periplasmic space

• Sec translocator

chloroplasts and mitochondria protein translocation similarities

1. needs energy from ATP and electrochemical gradient

2. occurs post-translationally

3. needs help from translocators in both membranes

4. SS clipped off

unique to chloroplasts in protein translocation

• can utilize GTP for NRG

• protein complexes: TOC and TIC

• only goes to the thylakoid if second SS is present, otherwise stays in stroma

• OXA used to embed in thylakoid membrane

chloroplast protein translocators

• outer: TOC

• inner: TIC

vesicle formation requirements

• protein cage

• this is formed during budding from smaller subunits to larger lattice but discarded BEFORE fusion (must shed so lipids can interact)

vesicle functions

• inner: concentrate proteins and cargo

• outer: shapes vesicle via lattice structure

categories of protein coats (3)

1. clathrin: endocytosis pathway

2. copI: retrieval pathway

3. copII: secretory pathway

pathways and direction (3)

1. endocytosis: membrane—>lysosomes

2. retrieval: ER to regions prior

3. secretory: ER—>golgi

clathrin

• form pits in membrane

• size can adjust to cargo carrying

• inner layer: adapter AP2 proteins

• outer layer: triskelions (more triskelions=bigger coat size)

clathrin cycle steps

1. adapters (AP2) bind —> concentrates cargo

2. triskelions bind to adapters and bend membrane. vesicle starts budding

3. protein DYNAMIN constricts and breaks membrane

4. vesicle is released

5. coat is shed via chaperones and ATP

6. lipids exposed for fusion

COPI/COPII coats layers

• inner: Sar1-GTP, Sec23, Sec24

• outer: Sec13/31

Sar1

• inactive when GDP-bound! found in cytosol

Sar1 cycle

1. membrane recruits Sar1GEF from membrane

2. Sar1GEF converts Sar1GDP into ACTIVE SAR1GTP

3. changes conformation

4. hydrophobic region can incorporate into membrane of ER

5. Sar1GTP must be in membrane for Sec23 and Sec24 to bind

6. Sec23 binds to Sar1

7. Sec24 binds to Sec23 and holds site for cargo receptor

8. Sec13/31 binds and vesicle budds off

9. vesicle pinched by protein DYNAMIN

10. coat is released through Sar1GTP hydrolysis

11. vesicle can dock!

vesicle docking steps

1. Rab effectors anchored to target interact with Rab proteins

2. tether made through RabGTP and Rab effector. must be in close proximity

3. SNARE proteins wind together and displace H2O

4. snares must then unwind (ATP dependent via NSF protein and ATP hydrolysis)

Rab proteins

• active in GTP-bound state

• specificity

Rab effectors

• proteins that protrude from target compartment membrane

• anchored to target and interacts with Rab proteins

SNARE proteins and types

• necessary for fusion

• V-snares: extend from vesicle

• t-snares: extend from target

• form trans-snare complex

• must connect!

secretory pathway (ER—>golgi)

• homotypic fusion forms vesicular tubular clusters (intermediate)

• transported by motor proteins along microtubules

• COPII

homotypic fusion

• biochemically similar vesicles form vesicular tubular clusters

• done via SNARE proteins

moving through the golgi

• has flattened stacks called cisternae

• CIS FACE: closer to ER

• TRANS FACE

• cargo moves from CIS to TRANS face

• covalent modifications in different cisternae

• 2 distinct pathways

golgi pathways (2)

1. cisternal maturation model

2. vesicular transport model

both exist but no correlation in differences

cisternal maturation model

• vesicular tubular cluster becomes new cis fac

• gets moved layer through layer

vesicular transport model

• fuses to cis face

• vesicles break off and individually carry cargo through layer

exocytosis

• delivery of lipids and proteins for membrane

• soluble proteins released

• 2 pathways

exocytosis pathways (2)

1. constitutive secretory: CONTINUAL

2. regulated secretory: vesicles wait in cytoplasm to be released (example: neurotransmitters)

resident proteins

• must stay in specific compartment, but may get trapped in vesicle

• creates need to recognize aka need for retrieval pathway

• ER loses its function without resident proteins

mechanisms to prevent losing resident proteins

1. make less favorable with coating

2. have ER retrieval sequences with specific SS

membrane proteins in retrieval pathway

bind directly to COPI

ER lumen proteins in retrieval pathway (water soluble)

KDEL sequences bind to receptors which bind to COPI

Endocytic pathways (3)

1. pinocytosis

2. receptor mediated

3. phagocytosis

steps along microtubule for endocytic pathway

1. endocytic vesicles

2. early endosomes

3. multivesicular bodies

4. late endosome (acidic)

5. lysosome (acidic)

6. recycled back

pinocytosis

cell binds in, liquid goes in

vesicle created

receptor mediated endocytosis

cargo bind to surface receptor to start budding of vesicle

phagocytosis

cell brings in larger items to break down

can fully enclose

receptor mediated endocytosis fates (3)

1. receptors go back to surface and recycled

2. transcytosis: receptor brought in one side and recycled out the other

3. degradation: delivered to lysosome

lysosome

• very small

• many enzymes to break down molecules

• acidic hydrolases

• HYDROLYTIC

• proton pumps drive H+ in lysosome and environment allows enzymes to be active

• plants have vacuoles with hydrolytic enzymes for digestion

hydrolytic

functions @ low pH

receptor mediated endocytosis steps

1. cholesterol packed inside LDLs

2. LDL binds to surface of cell wall

3. forms clathrin vesicle

4. coat sheds

5. fusion

6. early endosome

7. late endosome

8. lysosome

9. LDL recycled back into cell surface

phagocytosis

cell extends membrane out via building actin filaments to push while engulfing something for breakdown

autophagy

• lysosomal degradation

• can initiate cell death if mitochondria compromised

phagocytosis steps

1. INDUCTION: signal in cell. lipid vesicles start fusing around cargo

2. creates autophagosome - fully surrounded compartment

3. fuses with lysosome

4. digestion

5. recycles materials back

**IF MITOCHONDRIA DYSFUNCTIONAL: TAGGED WITH UBIQUITIN

synaptic vesicles

• creates vesicles to load with neurotransmitter via endocytosis

• vesicle waits to be recruited via transporter protein

glycolysis

• oxidizing and processing sugars

• 10 steps

• 3 phases

• per 1 molecule of glucose: 2 NADH, 2ATP, 2 pyruvate

• ANAEROBIC

glycolysis phases (3)

1. energy investment: convert glucose into fructose 1,6-biphosphate. uses 2ATP

2. cleavage: 2 3C sugars. no energy needed

3. NRG generation: 2 molecules pyruvate formed

processing carbs and fats (animal cell)

if not enough glucose in diet, use polysaccharide glycogen in liver and muscle cells

processing carbs and fats (plants)

use starch

steps of energy conversion (animal cell)

1. glycolysis

2. citric acid cycle

3. oxidative phosphorylation

citric acid cycle (animal cell)

• in matrix

• every turn = 3NADH, FADH2, ATP

• goal: make electron carriers for ETC

• reduce number of carbon by giving off CO2 as byproduct every turn

citric acid cycle steps

1. pyruvate enters mitochondria

2. acetylCoA combined with citrate once the coenzyme is gone

3. incorporated into oxaloacetate

oxidative phosphorylation (animal cell)

• inner mitochondrial membrane

• goal: make lots of high yield ATP

• AEROBIC ONLY

• need lots of electrons to drive process

• pass high energy electrons through complexes with increasing reduction potential, driving ADP—>ATP process

• NRG creates H+ flux driving ATP synthase pumps

• O2 final electron acceptor. combines with H+ to form water

ETC complexes (animal cell) (4)

1. NADH Dehydrogenase

2. Succinate Dehydrogenase

3. Cytochrome C Reductase

4. Cytochrome C oxidase

Complex I: NADH Dehydrogenase

• NADH drops of electrons, pumps H+

• L shaped

• hydrophobic membrane arm with alpha helix that mechanically triggers H+ pumps

• hydrophilic arm in matrix that contains iron sulfur clusters in which electrons jump off them to get to ubiquinone pool

• 4 protons from matrix per 1 NADH

• passes on electrons via fully reduced quinones

iron sulfur clusters

electrons jump off them to get to ubiquinone pool

Complex II: Succinate Dehydrogenase

• only entry point, does not pump H+

• electrons from FADH2

• usually bypassed

• ubiquinone proteins help move electrons from complexes