exam 2: lecture 6 Flashcards
how does a centrifuge work?
by spinning at a fast rate
Svedberg-30s
dependent on: density, size/volume, mass
-large, dense molecules move toward the bottom of the tube more quickly
method of isolating organelles: differential centrifugation
- begin with a uniform solute in tube
- low-speed: large solutes at the bottom
supernatant: liquid”above swimming” - do it again until the desired part is the pellet
why is medium isotonic? to make sure the organelles stay intact
method of isolating organelles: density gradient centrifugation
- add sample to tube of variable-density solution
2.run centrifuge. cell separated by DENSITY into distinct bands
- use needle to poke and withdraw specific band
Rough ER + Smooth ER
Rough:
part of the nuclear membrane
has ribosomes
internal: cisternae
*function: synthesis of proteins
Smooth:
no ribosomes
found in kidneys, liver
*function: synthesis of lipids (fat), steroid production+ detoxification
nucleus
nuclear envelope: double membranes; protects dna
pore: regulate ribosomes in & out
chromatin: coiled DNA
nucleolus: produce+assemble cell’s ribosomes
Smooth ER functions
found in the highest concentration in liver cells
- hydroxylation reactions: adding OH on aa (ETC)
- drug detoxification
3.glycogen catabolism: enzymes that help finish glycogen-> glucose (liver stores glycogen) - production of membranes
ex. phenylalanine–> tyrosine
what is the process of glycogen breakdown in liver?
- glycogen granule is broken off into individual units with P (attached 1st carbon) w/ glycogen phosphorylase
- phosphoglucomutase transfers P to the 6th carbon
why? to prevent glucose from immediately metabolizing by liver cells - glucose-6-phosphatase remove the P
- glucose transporter transport into blood from smooth ER
Steroid Biosynthesis in Smooth ER
-smooth er: the site of cholesterol & steroid hormone synthesis (adrenal glands,liver cells, sex hormones)
-large amount of smooth ER are found in cells that synthesize these
- SER also found associated with plastids in some plants and may be involved in phytohormone synthesis
What do cholesterol have in common with cortisol and steroid hormones?
they all share a 4-ring structure, but differ in the number of the arrangement of carbon side chains & hydroxyl
what is the protein that is in the committed step in cholesterol biosynthesis? and where is it found
HYdroxymethylglutaryl-CoA reductase (HMG-CoA reductase)
found in smooth ER of liver cells
-is targeted by satins (cholesterol-lowering drugs)
what does the ER play a central role in
the biosynthesis of membranes
with exceptions:
mitochondria: phosphatidylethanolamine
peroxisome: cholesterol
chloroplast: contain enzymes for chloroplast-specific lipids
Membrane biosynthesis: how are they synthesized
- fatty acids are synthesized in the cytoplasm -> incorporated into the ER membrane on the cytosolic side
how are they transferred to the lumenal side of the bilayer?
phospholipid translocators (flippases)
-this can lead to membrane asymmetry (depending on a specific translocator)
what are phospholipid exchange proteins?
convey specific phospholids to mitochondria, chloroplatsts, peroxisome
Rough ER functions
- has “chaperone” protein that ensures that newly made proteins fold correctly (final teriary structure is correct)
- glycosylated (glycosylation ) proteins for “post-translational modification”
- membrane proteins are transferred into membranes ( proteins will be attached to RER membrane first, then get transported to its wanted place
- send proteins to be sorted for transport into other organelles and for export to the golgi body”secretory pathway”
- if a protein fails to fold correctly, rough ER will direct it to be degraded (quality control-ER associated degradation (ERAD)
-occurs in cytosolic proteasomes
golgi functions
- packaging proteins for transport (inside)/export (move out entirely)
- sorting proteins for correct transport
- terminal glycosylation (finish process it started in RER)
- additional post-translational modification:
-phosphorylation
-sulfonation
-methylation (+ methyl)
What makes golgi different from the other organelles?
have polarity/ sidedness (an entry/ an exit)
cis: entrance (coatomer proteins)
media: between
trans: clathrin
what is core glycosylation?
occurs in RER
2 N-acetyl glucosamine
9 mannose
3 glucose
the attached sugars act as signaling devices for the ell to know how to treat the protein and where to send it–> zip codes
the process of core glycosylation
cytosol
start: dolichol is attached to P (hydrophobic)
1. another P is attached, along with N (P-P-N)
2. another N is attached (P-P-N-N)
3. 1st 5 (of 9) is attached (P-P-N-N-MMMMM)
*translocation: flippases from the cytosol to lumen
ER lumen
4. last 4 of M are attached (P-P-N-N-MMMMM-MMMM)
5. 3 G is added
6. the long chain is broken off and transferred to an aa (asparagine)
7. final processing: remove certain glucose and mannose units in the ER before the transfer of glycoprotein to the golgi
how does a flippase flip?
condition: two ATPs must be bound (energetic event)
1. substrate is bound to the flippase
2. ATP’s bound. the hydrophobic tail is twisted up.
3. the polar head is forced into the hydrophobic later-> nonpolar tail twisted down
4. ATP’s hydrolyzed as the polar head is pushed thru the opposite layer
What happens when terminal glycosylation in Golgi occurs
- nothing occurs to core sugar (get “high mannose” protein
- sugars are edited away to make the core small (glucose/mannose can be removed)
3.sugars are added to the core (N-acetyl-galactosamine, galactose, sialic acid
Compartmentalization of glycosylation: ER
-biosynthesis of core oligosaccharide for N-linked glycosylation
-attachment of core oligosaccharide to asparagine residues
-inital processing of core oligosaccharide
Compartmentalization of glycosylation: CGN
-attachment of N-acetylgalactosamine to serine. threonine
-1st step of phosphorylation of lysosomal proteins
-removal of mannose
-second step of phosphorylation of lysosomal proteins
Compartmentalization of glycosylation: Medial cisternae
-removal of mannose
-attachment of N-acetylglucosamine
-addition of galactose
-addition of sialic acid
Compartmentalization of glycosylation: TGN
addition of sialic acid
attachment of sulfate to tyrosine
what is anterograde transport?and the three types
forward: ER to Golgi
1. export (secretion)
2. storage
a. regulated (to be used)
b. constitutive ( go into storage)
3. organelles (lysosome)
what is retrograde transport?
backward: Golgi cisternae back to the ER
-allows the cell to balance the flow of lipids toward the membrane
-ensures a supply of material for forming vesicles
how is protein composition in the ER maintained?
prevent some proteins from escaping the Er
retrieve others from the golgi
Retention tag
what is the sequence: RXR (Arg-x-Arg)
=retention tag (keep the protein there;retains it)-will stay in the ER
-
tag must be masked to allow the assembled complex to leave the ER
ex. the N-methyl-D-aspartate (NMDA) receptor has the tag (important in mammalian neurotransmission
Retrieval tag
bring the proteins BACK
proteins returned from the Golgi to the ER
-short C-terminal sequences
ex. mammals: KDEL (Lys-Asp-Glu-Leu)/ KKXX
yeast: HDEL (His-Asp-Glu-Leu)
when a protein with this tag binds to a receptor, the receptor-ligand complex is packaged into a transport vesicle to return to the ER
How are Golgi complex proteins sorted
based on the lengths of their membrane-spanning domains (hydrophobic regions of Golgi proteins)
thickness increase: CGN (5nm)-> TGN (8nm)
*the proteins move from one compartment to another until the thickness exceeds the length of the domain
=this blocks further migration of particular membrane proteins
(protein is smaller than the domain)
how are proteins sorted in TGN?
soluble lysosomal enzymes undergo N-glycosylation followed by removal of glucose & mannose units
-mannose residues are phosphorylated in golgi-> form an oligosaccharide containing mannose-6-phosphate
*ensures delivery of lysosomal proteins to the lysosomes
steps:
1. RER: lysosomal enzyme is synthesized + carb is added
2. C-golgi: mannose is phosphorylated by 2 enzymes
3. T-golgi: mannose-6-phosphate bind s to recept + tagged enzymes are packaged in transport vesicles
4. Outside: low pH in late endosome (pre/immature lysosome) causes the receptor to dissociate from the enzyme
5. receptor is recycled
the process of phagocytosis/endocytosis
- membrane invaginates to form a pocket that hug macromolecules from outside
- pocket pinches off, closing off the material
3.close & form a vesicle
- vesicle separates from the membrane
exocytosis
- secretory vesicle approach membrane
- fusion of membrane
- rupture of cell membrane
- discharge of vesicle contents to the outside+ vesicle become part of membrane
what mediate fusion between vesicles & target membranes
SNARE hypothesis: states that sorting & targeting of vesicles involves 2 families of SNARE receptor
- v-SNAREs: found on vesicles
- t-SNARES: found on target membranes
-both are complementary molecules that allow recognition between vesicles & targets
two roles of clathrin in clathrin-dependent
a. clathrin-coated vesicles (CCV) selectively sort cargo at the cell membrane
b. the coating keeps the receptors/cargo from just floating away from the coated pit as vesicle is forming
adaptins: help the clathrin subunits to coat the vesicle as it forms
dynamic: motor protein that helps close up the vesicle once it has finished forming
the process of clathrin-dependent endocytosis ( receptor-mediated)
- binding: the ligand binds to the receptor-ligand complexes
- lateral diffusion: as the complexes move laterally, it is coated with clathrin w/ the help of adaptins
- invagination: the pit is invaginated and the vesicle is closed up with dynamic
- vesicle formation: the vesicle is now formed (clathrin is still coated)
- uncoating: the clathrin is uncoated (removed bc clathrin is not needed anymore as the vesicle is inside + do not want to the clathrin to mess u the chance of vesicle to bind)
6.fusion w/ early endosome and release of ligand
7a. transport to late endosome, then lysosome for digestion
7b. transport of receptors to cell surface for recycling
7c. transcytosis: transport vesicle out of cell (into small intestine into the blood)
what is the structure of clathrins?
triskelions
consists of terminal globular domain, heavy chain, light chain
(held at the vertex)
looks like a ball `
traffic thru the endomembrane sys.
- rough er
- transition vesicles to the golgi (cis-face)
- anterograde transport: ER to golgi
- secretory vesicles
a. constitutive: to be released
b. regulated: storage - secretory vesicles to be fused with early endosome
6 (early endosome)-> 8 (late endosome)-> lysosome
8(late endosome) fuses w the membrane of the golgi-> gets transported to the er= retrograde
note: no atp-> no secretions ( bc motor proteins use atp to move vesicles)
what is the third type of coated vesicles
caveolin: coat protein
caveolae: caveolin-coated vesicles
type of lipid raft rich in cholesterol & sphingolipids
involved in cholesterol uptake by cells ( help move cholesterol+ lipids around cells)
three types of coated vesicles
coatomer
clathrin
caveolae
what are the functions of lysosomes?
- digests nutrients for use by the cells
(acts a stomach: proteinates, nucleases, lipases, glycolaces) - in phagocytes: destroy foreign pathogens (bacteria)
- recycles damaged organelles+ structures no longer needed
- apoptosis: programmed cell-death (breaks open-> release all contents into cells)
autophagic vs. heterophagic lysosome
autophagic “self-eat”: promote cell survival by elimination of damaged organelles
heterophagic: digest food obtained from outside
how to protect the cytosol from being randomly breaking down by enzymes
- proenzymes: non-functional until exposed to the right environment (active site is closed off until low pH)
- only function @low pH (only function inside of lysosome)
lysosome role in cellular digestive processes
a. phagocytosis
b. endocytosis
c. autophagic vacoule forming around mitochondria
d.exocytosis releasing acid hydrolases to extracellular fluid
what are the conditions of apoptosis?
pH of cytosol has to dropped
1. development not occurring correctly
2. immunological reason: infected by a virus ( to reduce infection level)
3. kill potential cancerous cells
how is a plant vacuole a multifunctional organelle?
some are acidic-funtion as lysosomes
development: provacuole (endosome)
functions:
-mature to fill up to 90% of total vol
-maintain turgot pressure (osmotic pressure preventing the from collapsing by pushing water against wall)
-regulate cytosolic pH (using atp-dependent pumps)
-serve as a storage compartment
a.seed storage proteins
b. malate (in CAM plants)
c.anthocyanins (pigments)
d. soluble/ insoluble watse
peroxisomes: functions
location: liver + kidney tissues
structure: crystalline cores
size: peroxisome-> chloroplast-> mitochondria
lots due to high energy from lots of sunlight (matrix )
functions:
-detoxifying oxygen radicals (high energy ions-> radicals)
-detoxifying of harmful compounds (ethanol, methanol, formic acid, formaldehyde, nitrites, phenols)
-oxidation of fatty acids
-metabolic breakdown of nitrogen compounds (can make amonia; proteins, dna/rna)
-breakdow of “unusual substances” such as alkanes/ petroleum products (long chain carbons)
what are the reactive oxygen species dealt with in the peroxisome?
- oxygen single (O)
- superoxide free radical (O2)
O2- + O2- + 2H+ —super oxide dismutase—> H2O2 + O2
how does hydrogen peroxide metabolism work?
SOD generate H2O2 in peroxisomes
RH2 + O2 —-> R + H2O2
*it is being detoxified by catalase (obligate anarobes: will die when exposed to oxygen)
2H2O2–catalase—> O2 + 2H2O
*can also use peroxidase
R’H2 + H2O2 —peroxidase—> R’ + 2H2O
=same result: H2O2 is degraded while still inside peroxisome
where do the oxidation of fatty acids take place
animals:
long chain fatty acids are oxidized
once less than 16 carbons, oxidation occurs in mitochondria
plants/ yeast:
oxidized in peroxisomes
metabolisn of nitrogen-containing compounds
animals (not primates) require urate oxidase to oxidize urate
urate (toxic) + O2 —-> allantoin + H2O2
what does aminotransferases do
catalyze te transfer of amino groups to alpha- ketoacids in the degradation & synthesis of aa
catabolism of unusal substances
d-aa are rare where there is no degrative pathways, except in perisomes
in some cells, peroxisomes have enzymes that can break down xenobiotics (compounds foreign to living organisms)
-including alkanes ( short chain hydrocarbons)
