Chapter 5 Cell bio Notes

endosymbiotic theory

theory that eukaryotic cells formed from a symbiosis among several different prokaryotic organisms

evidence for the endosymbiotic theory

Mitochondria and chloroplasts possess their own DNA similar to prokaryotes; ribosomes resemble that of prokaryotes;

they reproduce independently of the cell using binary fission

-use of porins

chemical components found in mitochondria and also bacteria cells

cardiolipin

-this supports the idea that bacteria is the origin for these organelles

-also use of protein translation with N-formyl methionine just like bacteria where eukaryotes just use unmodified methionine

chloroplasts

makes ATP but all of that ATP is used in the chloroplasts.

All energy for the cell is from the mitochondria

first endosymbiotic event

ancestral eukaryote consumed aerobic bacteria that evolved into mitochondria

second endosymbiotic event

the early eukaryote consumed photosynthetic bacteria that evolved into chloroplasts

carboxysomes

homologous

same origin different thing

analogous

same thing different origin

higher the sedimentation coefficient

the higher the weight

examples of genes in the mitochondrial genomes

-components of the transcription/translation machinery

-components of the electron transport chain (complex I,II,IV)

-specific components of the ATP synthase

complex 2 proteins are imported

chloroplast genome

encodes for chloroplast genes, only about 120 genes

what is missing from the endosymbiotic organelle genomes?

Genes were either lost over time since they were not important

and they were transferred to the nucleus instead

- , the majority of proteins found in mitochondria and chloroplasts are now encoded by nuclear genes due to mutations

mitochondria thing

2 lipid bilayers

-4 photosystems and the ATPase

which photosystem does not contain ribosomes?

photosystem 2 since they are imported

how do we import proteins into the mitochondria and chloroplasts?

via TIC_TOC and TIM-TOM

-use of a signal molecule and a receptor for transport as well as translocate machinery

protein import into the mitochondria

TOM complex hold the receptors on the outer membrane

TIM 23 vs TIM 22

TIM 23

-contains a hydrophobic alpha helical extension that allows it to span both of the membranes

TIM 22 complex

just sits on the inner m membrane, does not go through both membranes

soluble protein targeting to the mitochondria

an amino acid targeting sequence will be used to identify proteins for the mitochondria

the targeting sequence (presequence) for mitochondrial targeting must:

1. be on the N terminus of the protein so it exits first from the ribosome

2.be the specific amino acid that is used for mitochondrial targeting

3. shape itself into an alpha helix that will become a binding pocket for SRP

-one side of the helix is polar and the other is nonpolar

steps for the import of mitochondrial matrix proteins

1. the entire protein is synthesized in the cytoplasm

2. chaperones like HSP 70 maintain an unfolded protein state until it reaches the mitochondria

3.the protein moves through the outer and inner membrane via TOM and TIM (tom is outer tim is inner) using energy from ATP hydrolysis

4. In the mitochondrial matrix, the targeting sequence is removed by MPP and the protein is folded and processed

protein targeting to specifically the outer mitochondrial membrane

2 methods

1. Beta barrels create pores that are recognized by TOM and inserted into the outer membrane by SAM (sorting/ assembling machinery)

2. Using MIM protein complex

MIM protein complex

promotes the insertion of proteins with N or C terminal anchors

-can do this by either interacting with TOM or using free MIM complexes to do the work

protein import to specifically the inner mitochondrial membrane or intermembrane space

-use of two signal sequences. The first does exactly what is needed for the outer mitochondrial membrane and cleaved off.

-the second will direct the protein into the inner membrane using the OXA dependent pathway seen in the mitochondrial matrix transport flashcard

translocation arrest in TIM

if the hydrophobic sequence after the target signal accidentally binds to TIM23, translocation will halt.

-the rest of the protein is pulled through the TOM into the outer membrane and the hydrophobic portion is stuck in the inner membrane

insertion of multipass inner membrane proteins (G proteins)

done by TIM22 instead of 23

protein targeting to the chloroplast stroma (plants)

-the targeting sequence is called the transit peptide, is a different sequence than for animals but is also an N terminal sequence

-the transit peptide is recognized by the chloroplast import receptor

-use of TOC and TIC via energy use

-signal is cleaved once the protein enters the stroma by the SPP

differences between mitochondria and chloroplasts

mitochondria's generate energy via ATP

chloroplasts are creating mainly sugars through photosynthesis, very little ATP because it can't leave the membrane due to lack of transporters

what are the three ways to produce ATP?

1. Chemiosmotic coupling:

a. oxidative phosphorylation by the mitochondria

b. photophosphorylation in the chloroplast

3. Substrate level phosphorylation via glycolysis

how does chemiosmotic coupling work?

a protein pump uses an energy source to push protons against the gradient of ETC

-this energy is converted to potential energy and can be used to drive the phosphorylation of ADP to ATP using AT synthase complex

structure of ATP synthase

-The F0 region is the base

-the F1 region is the round head

an axel connects them and moves the head of F1 by a long protein called the stator

-a kink in the membrane occurs due to two ATP synthases dimerizing

F0

is the base embedded into the membrane. Protons flow through here to turn the axle, creating ATP

why is chemiosmotic coupling different in mitochondrias and chloroplasts?

it is believed that they originate from different endosymbiotic events

-chloroplasts exhibit inwards projections of their membranes and an extra set of membranes not seen in the mitochondria

mitochondrial chemiosmotic coupling

a gradient occurs across the inner membrane

-as proteins are pumped out, the PH goes up and the membrane becomes basic

-this PH change does not affect anything else in the organelle which is good

chloroplast chemiosmotic coupling

done on the thylakoid membrane

-protons are pumped into the thylakoid lumen, decreasing pH which is more significant here

-this membrane is permeable to Cl and Mg, making it nonelectrical so energy is only from the difference in chemical concentration

how do mitochondria and chloroplast work together?

Chloroplasts make the glucose and oxygen from CO2 and water, and mitochondria take the glucose and oxygen and turn it into energy and CO2.

what did fluorescence micrography discover about mitochondria?

that they are constantly moving and adapting and dividing

glycolysis occurs in

cytosol

pyruvate is converted to

Acetyl CoA in the mitochondria

acetyl coa is fed into TCA cycle creating

NADH and FADH2

electron carriers in the mitochondria

what membrane is the ETC and ATP synthase attached to ?

the inner membrane

other things the mitochondria does

1. ionic regulation (calcium homeostasis)

2. cell signaling

3. Heat generation

4. biosynthetic pathways

5. apoptosis

etioplast

precursors of chlorophyll

chromoplasts

pigment synthesis and storage

leucoplasts

fatty acid and amino acid synthesis

amyloplasts

starch and sugar storage

elaioplasts

store lipids and oils

proteinoplasts

sites of enzyme (Protein) activity

in seeds, all plastids are

proplastid. As the seed germinates, the shoots and above ground tissue will develop into etioplasts before they reach the light. Once they break through the soil and are exposed to light, the etioplasts convert into chloroplasts. Below-ground tissue, like roots, will develop into unpigmented leucoplasts, which may then convert into storage plastids such as amyloplasts (to store starch), elaioplasts (to store lipids), or proteinoplasts (to store protein). Chloroplasts can also convert into colored plastids known as chromoplasts. This process can be seen when fruits ripen and change color or when the fall leaves change color before they drop

in order for chloroplasts to keep up photosynthesis, they must

fix atmospheric carbon, reduce it, and turn into carbs the cell can use

-this occurs by pumping proteins between the thylakoid lumen and the stroma producing ATP and NADH

-it must be put through the Calvin cycle to become sugars since it cannot release ATP

other responsibilities of chloroplasts

-production of fatty acids, amino acids, and vitamins

-nitrogen metabolism

-stress response

-starch storage

-retrograde signaling

mitochondrial membrane potential

occurs due to the pumping of protons on the etc

-provides energy to power ATPase

-since the matrix is negative an accumulation of positive chemicals will accumulate, which can be measured

techniques used to measure MMP

JC-1, TMRE, and DiOC6

-fluorescent dyes that accumulate in the mitochondria and emit a light

JC-1 Assay

use of a lipophilic cationic dye

-in healthy cells, it will accumulate and exhibit a red color

-in unhealthy cells, it will not enter the mitochondria, and the image shows up as green

mitochondrial oxygen consumption rate (OCR) analysis

allows you to determine oxygen consumption and H+ release in living cells

mitochondrial inhibitors

-rotenone

-antimycin A

-oligomycin

-FCCP

rotenone

Blocks NADH ubiquinone reductase by inhibiting transfer from complex 1 to ubiquinone

-leads to decreased potential and cell death

antimycin A

inhibits the ETC by inhibiting complex III

-decreases MMP

oligomycin

inhibits ATP synthase by binding to it, blocking proton flow

-also inhibits oxygen uptake

FCCP

uncouples mitochondrial oxidative phosphorylation

-allows us to study mitochondrial function but leads to apoptosis

cyanide toxicity

cyanide binds to the iron atom on a heme on complex 4, preventing cells from using it for ATP production

-oxygen deprivation occurs and cell death happens