Exam 4: Organelle Biogenesis (Bolded from original ppt + review slide information)

How large are peroxisomes typically?

0.2 microns in diameter

How many membranes does the peroxisome have?

1

Each AP complex contains what?

1 copy each of 4 different adapter subunits

Schematic of receptor-mediated endocytic pathways.

1. LDL particle binds to LDL receptor on plasma membrane 2. LDL/LDL receptor complex internalized by clathrin-coated pit pinches off forming a coated vesicle 3. Clathrin coat depolymerizes. Vesicle with cargo fuses with early endosome. Recycling of some cargo occurs via early (fast) and recycling (slow) endosomes 4. early endosome matures to late endosome (multi-vesicluar body, MVB). Moves toward perinuclear region 5. Late endosome accepts cargo (lysosome hydrolases) from Golgi apparatus 6. late endosome fuses with lysosome delivering cargo from plasma membrane/Golgi

What is the first step in the NES (nuclear export signal) mechanism?

1. formation of exportin 1/Ran-GTP/cargo complex transported through NPC

What is the NLS mechanism? What are the steps?

1. in cytoplasm, importin alpha and beta form complex with cargo protein 2. importin B interacts with NPC for translocation 3. RanGEF converts Ran GDP to Ran GTP; RanGTP interacts with importin B dissociating complex 4. the importin alpha and importin B/Ran-GTP complex are translocated to the cytoplasm 5. in the cytoplasm, RanGAP convertes RanGTP to RanGDP, dissociating from the importin B and reinitiating the cycle.

How do mitochondria import proteins?

1. precursor proteins synthesized on cytosolic ribosomes are maintained in the UNFOLDED STATE by bound chaperones (Hsc70). 2. precursor protein binds to import receptor 3. precursor protein transferred to general import pore 4. precursor protein moves through channel and adjacent channel in inner membrane (for matrix proteins) 5. binding of the precursor protein by Hsc70 and subsequent ATP hydrolysis drives import 6. targeting sequence cleaved and protein folded.

How many base pairs are in human mtDNA?

16, 560 bp

How many membranes does the nucleus have?

2 outer and inner membranes outer (nuclear envelope) is continuous with the ER - has docking proteins mediating ribosomes binding to outer leaflet Inner nuclear membrane is supported by lamins

How many membranes do mitochondria have?

2 membranes outer = 50% protein/50% lipid inner = 80% protein/20% lipid

Human mtDNA codes for what?

2 rRNAs, 22 tRNAs, 13 polypetides

What makes the NES mechanism keep going?

2 things the concentration gradient between tri-molecular complex between the nucleus and the cytoplasm - this is torn down into 3 molecules immediately, so you always have a high concentration of Ran, exportin-1, and cargo constantly in the nucleus - so it always wants to go there. RanGEF is always in the nucleus and RanGAP is always in the cytoplasm.

What is the life expectancy of infantile Tay Sachs?

3 years old

When the clathrin polymerizes on the donor membrane, it does so in association with what?

AP (adaptor protein) complexes

What is the acetyl-CoA in mitochondria used for?

ATP production

What is the 3rd step in the NES mechanism?

Cargo remains in cytoplasm exportin-1 and Ran-GDP translocate into the nucleus

What will you find inside the inner membrane (matrix)?

DNA, ribosomes, chromosomes granules where oxidative phosphorylation occurs. beta oxidation occurs here too.

What are the main recipients of incoming endocytic vesicles from the plasma membrane?

EEs (early endosomes)

What is another lysosomal storage disease?

I-cell disease (inclusion) aka mucolipidosis II

Describe how mitochondria uptake mid and short chain fatty acids and what happens after uptake.

In the cytoplasm, mid or short cahin fatty acids with covalently linked acetyl-CoA give a fatty acid-CoA. This will then find its way past the outer membrane into a carnitine transporter in the inner membrane. It then exchanges an acetyl-CoA moiety for a carnitine. It locks to the transporter and as soon as the fatty acid-carnitine complex gets into the inner matrix, it again exchanges the carnitine for an acetyl-CoA moiety. The beta oxidation machinery recognizes this and beta oxidation then occurs. Now it takes the acetyl-CoA, which is used in the citric acid cycle. These enzymes are ultimately used in the oxidative phosphorylation to generate ATP. Glycolysis can also lead to the formation of ATP - pyruvate converts to acetyl-CoA.

What is the key difference between the peroxisome and the mitochondria?

In the peroxisome, we fold the protein - we don't cleave the peroxisomal targeting signal. The protein goes through the membrane folded. In the mitochondria, the protein goes through the membrane unfolded and we cleave the targeting signal.

How are lysosomal enzymes delivered to lysosomes?

M6P tag! 1. synthesis in ER 2. enzymes requiring M6P ligand move to cis-golgi, where M6P acquisition occurs 3. interaction of enzymes with M6P receptors (budding from trans-golgi) - once it gets to trans golgi, an M6P receptor recognizes it, grabs it and places it in a clathrin-coated vesicle. 4. receptor-protein complex moves to late endosome, where dissociation occurs 5. receptors are recycled to plasma membrane or back to golgi 6. delivery to lysosome

How do you get kidney stones?

Mix up in PH1 - a pseudo-targeting signal was made and because the protein was defective anyway - it could not fold. The cell thought this must belong in the mitochondria and sent it there - thus kidney stones

Describe the steps involved in the the import of peroxisomal matrix proteins. "Stuffing a peroxisome"

Note: peroxisomal matrix protein to be imported translated on cytosolic ribosome 1. Ser-Lys-Leu-COOH import signal PTS1 of cargo binds to Pex5 receptor 2. Pex5-cargo docks at membrane 3. Pex5-cargo translocates through the membrane (folded state) and dissociates 4. export of Pex5 (PTS1 (tail of an enzyme) is NOT cleaved) 5a. Recycling of Pex5 - monoubiquitination of Pex5 by Pex 2, Pex10, Pex 12 and either Pex4 or cytosolic ubcH5a/b/c, in humans 5b. polyubiquitination by Pex2, 10 and ubc4/5 (yeast) or ubiquitin-conjugating enzymes E2D2 (humans) for proteasome degradation 6a. Pex5 recycling from the peroxisonme membrane back to the cytosol by the action of AAA ATPases (Pex1,6,26 - triple ATPases) and ATP hydrolysis. Theses AAA ATPases are a family of proteins that couple the eneryg of ATP hydrolysis to disassembly of protein complexes 6b. Degradation of a receptor that is blocked from recycling 7. deubiquitination of the receptor before the next round of import. The squiggly line on Pex5 denotes its disordered N-terminal segment.

Peroxisome biogenesis? How are peroxisomes synthesized?

PMPs (peroxisomal membrane proteins) are imported into the ER, are sorted into ER pre-peroxisomal compartments and bud out in a Pex3/Pex-19 dependent manner to form pre-peroxisomal vesicles. These vesicles can form mature peroxisomes after fusion, dpendent on Pex1/Pex6 and matrix protein import (de novo pathway). Alternatively, the pre-peroxisomal vesicles fuse with divided peroxisomes generated from pre-existing mature peroxisomes. Peroxisome division requires Pex11 and a specific set of DRPs (dynamin-related proteins)

What is included in the endomembrane system?

RER, SER, golgi functionally interconnected and are almost all membrane

What is the reaction of B-oxidation?

RH2 + O2 --> R + H2O2 H2O2 + R'H2 --(catalase)--> R' + 2 H2O (RH2 = substrate reduced) H2O2 = extremely toxic to life - must get rid of it.

Describe what happens in late maturation of LEs.

Rab switch formation of ILVs acidification change in size and morphology (tubular extensions present on EEs are lost and the endosomes acquire a round or oval shape and grow in size) loss of recycling with plasma membrane - recycling receptors are lost from the organelle and recycling of membrane and fluid to the cell surface stops gain of lysosomal hydrolases and membrane proteins - these lysosomal components are transported mainly from the TGN a switch in cytoplasmic motility - the endosoms associate with a new set of MT-dependent motors that allow them to move into the perinuclear region of the cell

What is exportin-1 bound to?

Ran, the GTPase in the GTP state (activates the protein)

What is the 2nd step in the NES mechanism?

RanGAP hydrolyzes Ran-GTP to Ran-GDP, which dissociates the complex. (off state) lets go of the cargo and exportin-1

What are GTPase switch proteins?

RanGDP = 3D conformation = OFF when cell wants to activate GEF grabs the GDP and takes it off and replaces it with a GTP, then Ran changes shape and is ON. then GAP recognizes RanGTP and hydrolyzes it back to RanGDP. (not a swap this time) there are more GTP switches than just Ran..there is Rab, Ras, Rho, etc. hundreds. Ran often goes wrong in cancer (mutation makes it stay on - no hydrolysis back to GDP) - great targets for cancer too.

What is the 4th step in the NES mechanism?

RanGEF( nucleotide exchange factor) converts RanGDP to RanGTP and the cycle starts again

What is an example of a lysosomal storage disorder?

Tay Sachs disease

Three different AP complexes are known (AP1, AP2, AP3), each with four subunits of what?

Three different AP complexes are known (AP1, AP2, AP3), each with four subunits of different though related proteins. GGA is a fourth type of AP complex

What does the triskelion form?

a cage that looks like a soccer ball around the membrane that is invaginating. This will trap the LDL receptor and its particle it is holding onto. Assembly particles allow that to happen by binding to the clathrin-binding site on the heavy chain. Those particles will bind to the LDL receptor and to clathrin.

What is Tay Sachs disease caused by? What is Tay Sachs disease?

a mutation in the Hex A gene this mutation prevents the breakdown of ganglioside GM2 in the lysosome this results in progressive destruction of nerve cells in the brain and spinal cord

The developing LE contain what?

a selected subset of endocytosed cargo from the EE, which they combine en route with newly synthesized lysosomal hydrolases

What is a key reaction that occurs in cells (in peroxisomes)?

beta-oxidation ultimately the basis of a lot of things - particularly the production of energy/ATP. uses fatty acids to produce acetyl CoA (knocks it down 2 carbons at a time)

What is the shape of mtDNA?

circular

What are the 3 different stages of the golgi?

cis stage medial stage trans golgi network stage. glycosylated specificially with a distinct set of enzymes at the cis, medial and trans stage. In the trans stage, they will be placed into new transport vesicles and transported by Clap out of the cell or to a lysosome.

Why does the complex go toward the cytoplasm?

concentration gradient

What is the inner membrane of mitochondria organized into?

cristae

What is I-cell disease (inclusion) aka mucolipidosis II?

defect in targeting lysosomal enzyme enzyme is fine, but it can't get to the lysosome Causes abnormalities of face/skull, growth delays, mental retardation.

What is the function of the lysosome?

degradation and recycling where things go to die and then get recycled into the cell into its basic parts

Patients with the infantile form of Tay Sachs will suffer from what two major symptoms by age 2?

dementia and blindness

What is the function of the SER?

detoxicification and steroid synthesis tubular, lacks ribosomes synthesis of fatty acids/phospholipids (steroid synth) SER enzymes detoxify hydrophobic compounds

What do the mtDNA polypeptides code for?

enzyme subunits involved in electron transport and ATP synthesis

What is the NES bound by?

exportin-1

As the EEs are moving toward the perinuclear space along MTs, the nascent LE are what?

formed inheriting the vacuolar domain of the EE network

After proteins are transcribed or stranslated by the ribosomes, where do they go?

get pulled into the cisternae of the golgi which is continuous with the nuclear outer membrane

What kind of reactions do LEs undergo?

homotypic fusion reactions (with other LEs), grow in size and acquire more ILVs

What does the lysosome contain a large number of?

hydrolases: - proteases - phosphatases - lipases - phospholipases and more

Generally where does cargo destine for lysosomes remain?

in the EE vacuole, whereas cargo to be recycle enters the tubular network

In the EE vacuole intraluminal vesicles (ILVs) are present. How do they form?

inward budding of the EE limiting membrane

How do ILVs form?

inward-buddin gof the limiting membrane forms ILVs containing cargo targeted for lysosomal degradation (super acidic in here too - lumenal pH drops from above pH 6 to pH 6-4.9)

What is the pH of the early endosome as compared to the cytoplasmic pH? What does this allow for?

it is LOWER than cytoplasmic pH (6.2-6.5) allows for sorting to begin aka, like with LDL.... the receptor will bind most tightly around pH 7 to the LDL particle - once it gets into the organelles where pH is getting lower, it will let go.

Where does destruction of an LDL particle happen?

mature late endosome fuse with lysosome and that is where destruction occurs

Which organelle is the ATP source?

mitochondria

EEs are heterogeneous in terms of what?

morphology, localization, composition, function

Does mtDNA have introns?

no

Can mitochondria take up long chain fatty acids?

no, but it can take up mid and short chain fatty acids

Does the nucleolus have a membrane?

no, it is a transient structure.

? ?? How do nucleoli form?

nucleolar organizer region - part of the nucleolus pars fibrosa and granulosum, but the nucleolar organizer gets everything started

Where are most proteins localized in the mitochondria synthesized?

on cytosolic ribosomes and imported into the organelle about 900 of these

Where are all proteins encoded by mtDNA synthesized?

on mitochondrial ribosomes

What is the key function of peroxisomes?

oxidation of organic substrates (very long-chain fatty acids) other functions: biosynthesis of plasmalogen phospholipids reduction of reactive oxygen species (ROS)

What is the pH in the lumen of lysosomes? What is this pH driven by?

pH = 5, driven by ATP pump optimum pH = 5

How is the oxidation in mitochondria different than in peroxisomes??

peroxisome takes a fatty acid down to an acetyl-CoA and a shorter fatty acid. The acetyl-CoA in the peroxisome will be used in a variety of biochemical reactions Acetyl-CoA in mitochondria is used in ATP production

What is are the functions of mitochondria?

production of ATP (main cell energy source) via oxidative phosphorylation - fatty acids (beta oxidation followed by oxidative phosphorylation) - contribute to regulation of cytosolic calcium levels - mediate apoptosis - participates in steroid hormone synthesis - in liver cells, mediates detoxification of ammonia

What is the function of the golgi apparatus?

protein and membrane processing/sorting; glycosylation of proteins and lipids glycosylation/assembly of proteoglycans - tag with M6P (mannose-6-phosphate) sorting for transport has 3 different stages

What is clathrin/clathrin-coated pits?

protein with a heavy and a light chain and forms a triskelion. Assembly particles: - composed of adaptor proteins - bind to heavy chain to promote polymerization - bind cytosolic-face membrane proteins to target bud. - bind to cytosolic portion of LDL receptor.

Where do early endosomes "patrol"

right under the plasma membrane., looking for cargo to tak into the endocytotic pathway.

What is the function of the RER?

site of protein and membrane synthesis - plasma membrane proteins - secretory proteins - membranes core glycosylation disulfide bond formation polypeptide folding protein subunit assembly (has ribosomes)

What is the function of EEs?

sore cargo for recycling to the PM, the TGN, or move it forward to the late endosome (LE)

At what point is there no more recycling back to the plasma membrane?

when the late endosome takes on lysosomal hydrolases. unlike in the early endosome.

By binding to the cytosolic face of the membrane proteins, the AP complex determines what?

which cargo proteins are specifically included in (or excluded from) a budding transport vesicle.

How does the mitochondria produce ATP?

takes a fatty acid and breaks it down into ATP

What does a peroxisome do with a fatty acid?

takes it down to acetyl-CoA and a shorter fatty acid. this acetyl-CoA will be used in a variety of biochemical reactions.

An association between the globular domain at the end of the clathrin heavy chain and one subunit of the AP complex both promote what?

the co-assembly of the clathrin triskelions with the AP complexes and adds to the stability of the completed vesicle coat.

Most EEs are relatively small and patrol where?

the peripheral cytoplasm close to the plasma membrane through saltatory movement along microtubules

What is the problem with the nuclear pore complex?

the vast majority of cargo that needs to cross the nucleus is bigger than can fit through the nuclear pore complex

What happens to a lysosome that bursts or releases?

they are neutralized since they operate at a more acidic pH than the cell

What do the two strands of mtDNA code for?

they code for different products strands = H and L

Early endosomes have what kind of shape?

unique shape/tubular endosomal network

What do EEs contain?

vacuolar part and a tubular network

Does the genetic code use in animal and fungal mitochondria differ from the standard code used in all prokaryotic and eukaryotic nuclear genes?

yes, it even differs in mitochondria from different species