4: Endomembranes Flashcards
Endomembrane system
-interconnected system of cytoplasmic membranes
- includes:
- ER
- golgi
- endosomes
- lysosomes
Movement out of cell
- biosynthetic or secretory pathways
- ER —> Golgi —> various locations
Movement into cell
- endocytic pathways
- plasma membrane —> endosomes —> lysosomes
ER
- continuous with outer nuclear membrane
- rough ER folds into sheets
- smooth ER is more tubular branched shape (coral)
- inside of ER called lumen
Smooth ER
- not involved in protein synthesis
- functions:
- Drug detoxification: enzymes add -OH to hydrophobic compounds to become water soluble and easier to excrete
- Carbohydrate metabolism: glycogen -> glucose (maintain blood glucose
- Calcium storage: important for muscle cells
- Steroid biosynthesis: cholesterol gets made
- lots of smooth ER in liver
Rough ER
- has ribosomes
- important in synthesizing proteins in secretory pathways
- first step of glycosylation/folding
-site for protein processing/modification/quality control
Ribosomes
- large (60S) and small (40S) subunits in eukaryotes
- made of ribosomal RNA (rRNA) and protein
-function: protein synthesis
Where are ribosomes found in the cell
- mitochondria and chloroplasts
- attached to membranes
- free in cystol
-ribosomes found in cystol and attached to membranes are THE SAME - structurally identical
Free ribosomes vs membrane-bound ribosomes
- Free:
- proteins destined to remain in cytosol
- proteins that go into nucleus
- proteins that go into mitochondria
- eg. Actin/tubulin
- Membrane bound
- proteins that are to be secreted or reside inside organelles in the endomembrane system (ER/golgi/lysosomes)
- protein/peptide hormones
- proteins that are integral membrane proteins of organelles in the endomembrane system
- proteins that are to be secreted or reside inside organelles in the endomembrane system (ER/golgi/lysosomes)
-ALL proteins start off as free ribosomes in cytoplasm, and then go to ER if needed/destined
How does a ribosome know to go to ER or not
- Gunter Blobel 1999
- Signal hypothesis:
- All ribosomes are the same
- An amino acid signal on a new protein directs the growing polypeptide and ribosome to the ER
- That protein will be fed into the ER lumen as it is translated (co-translationally)
Signal mechanism for cotranslational import
- A Signal Recognition Particle (SRP) temporarily binds to the signal sequence
- SRP = 6 proteins and a piece of RNA
- once SRP binds, translation stops
- The entire complex binds to a translocon in the ER membrane
- GTP is hydrolyzed, SRP leaves, and the new polypeptide is fed through the pore in the translocon.
- signal sequence is cleaved by signal peptidase
- Completed polypeptide released inside the ER lumen
- ribosome leaves
- pore closes
What is a translocon
- SRP receptor = binds SRP
- ribosome receptor = binds ribosome
- pore - channel (new protein feeds through)
- signal peptidase = enzyme that cuts off signal sequence AAs
Experimental evidence that Blobel was right
- experiment 1 = subcellular fractionation/cell-free systems
- ie. squish a bunch of cells, spin them at different speeds to separate different cell components
-experiment 2= modify a cytosolic protein by adding a signal sequence to its end and watch where it goes
How transmembrane proteins with C-terminus in cytosol get made
- Signal sequence targets polypeptide to translocon
- Stop transfer sequence halts translocation
- signal sequence cleaved by signal peptidase
- Protein released laterally into ER membrane
- N terminus is in ER lumen
- C terminus in cytosol
How a transmembrane protein with N-terminus in cytosol gets made
- The start-transfer sequence in the MIDDLE of polypeptide directs complex to ER
- start-transfer sequence locks the polypeptide in the translocon in the correct orientation
- Protein continues translocation until the c-terminus moves through the translocon
- Protein released laterally into ER membrane
How multipass transmembrane proteins get made
- Strat-transfer sequence starts polypeptide transfer
- Protein continues translocation until stop-transfer sequence encountered
- Portion of protein released laterally into ER
- next start-transfer sequence repeats process to initiate second transmembrane region
Import into mitochondrial matrix
- example of post translational import
- proteins first made in cytosol by free ribosomes, then imported to mitochondria if they have TRANSIT sequence
- TOM=translocase in OUTER membrane
- TIM=translocase in INNER membrane
- Hsp70 binds to new protein so it stays mostly unfolded
- New protein goes to mitochondrion and transit sequence binds to the receptor part of TOM
- Hsp70 goes away and new protein moves through TOM and TIM
- Transit peptidase cuts off transit sequence
- Mitochondrial Hsp70 will help pull the protein through the matrix by binding and not allowing protein to go backwards
-various types of TIMs: some allow protein to go straight through and other will allow protein to go sideways and stay in inner membrane
Golgi apparatus
- camillo golgi 1906
- proteins and membranes can travel from ER to golgi in vesicles
-compartments of golgi called CISTERNAE
Golgi functions
- the “processing plant” of the cell
- further protein modifications (glycosylation) and trafficking
- 3 parts:
- CGN
- medial cisternae
- TGN
Why separate compartments?
-different enzymes reside in different compartments to ensure processing happens in an organized, sequential manner
How does cargo (i.e.) proteins move through the golgi
- conflicting views!!!
1. Stationary cisternae model
VS
- Cisternal maturation model
Stationary cisternae model
- cisternae are stable compartments
- cargo gets shipped from compartment to compartment and eventually leaves through the trans face
Cisternal maturation model
- more widely accepted
- cis cisternae formed by fusion of vesicles coming from ER
- cisternae mature as they move from the cis face to trans face
- cargo stays in the same compartment and the golgi-resident enzymes are shipped backwards by retrograde transport to return to the “home” compartment
Evidence of cisternal maturation model part 1
- cargo protein is labelled
- it seems to only be found in cisternae, never in vesicles
- therefore can only get there because of maturation, NOT being transported
Evidence of cisternal maturation model part 2
- golgi-resident protein labelled
- see it in cisternae AND vesicles, which are moving in the retrograde directions
Evidence of cisternal maturation model part 3
- if you use a mutant cells that doesnt allow vesicles to leave the ER, then eventually the golgi will disappear completely
- if the cell resumes sending out vesicles the golgi will appear again
Post-translational modifications
- Proteolytic cleavage
- Glycosylation
- N linked
- O linked
Membrane carbohydrates (glycoproteins)
- N-linked: sugar attaches via ASPARAGINE
- O-linked: sugar attached via SERINE or THREONINE
- ALL carbohydrate side chains added in the ER start off with a common core oligosaccharide
- core oligosaccharide grows on a lipid called DOLICHOL PHOSPHATE
How N-lined oligosaccharides are made in ER
- a core oligosaccharide is made in the ER before it gets moved onto a protein
- carbohydrate chain will grow on DOLICHOL PHOSPHATE in ER membrane
- GLYCOSYLTRANSFERASES add monosaccharides to dolichol on CYTOPLASMIC side
- a flippase will flip dolichol and attached sugar chain so that it faces the lumen of ER
- more sugars get added
- Oligosaccharide protein transferase will take the core oligosaccharide off dolichol and add it to asparagine
Quality control
Process of N-linked glycosylation is important in one type of quality control - making sure the proteins are folded properly before leaving the ER
Process of quality control
- after the core oligosaccharide is added to the protein, 2 of 3 glucoses are removed
- CLANEXIN will bind to protein with one glucose on core oligosaccharide to give it time to fold
- Once folded, glucosidase II will remove 3rd glucose
- If NOT folded correctly, a glycosyltransferase called UGGT will ass a glucose back one
- Calnexin will recognize it, bind, and give it another chance to fold
- If irreparable, the protein will be recognized by a transporter and fed back out to the cytoplasm via reverse translocation to be destroyed by a proteosome
Proteasomes
- barrel-shaped protein degrading machines
- series of ring like subunits with a cap on either end
- only digests proteins that have been tagged for destruction via UBIQUITINS
Ubiquination
- ubiquitin is a small peptide (8.5kDa)
- 3 enzymes required to add ubiquitin
- E1 and E2 are ubiquitin carriers
- E3 is ubiquitin ligase (recognize misfolded proteins and transfer ubiquitins from E1 and E2 to misfolded protein
- misfolded protein is now POLYUBIQUITINATED and will bind to cap of proteasome
- proteases in proteasome break down proteins into amino acids
Unfolded protein response
- normally BiP (molecular chaperone) binds to unfolded proteins in lumen, some BiP says in ER membrane bound to sensors
- if there are too many unfolded proteins, BiP will be recruited from membrane
- protein sensors do 2 things once abandoned by BiP:
- Reduce overall protein synthesis in cell
- Increase synthesis of helpful proteins
Reduce overall synthesis response
-sensors add a PO4 to elF2a, which will bind to small subunit of ribosome and reduce synthesis
Increase synthesis of chaperones response
-a sensor protein is cleaved and acts as a transcription factor to make more proteins that alleviate ER stress
Retention and retrieval sequences
- Arg-any AA-Arg tags are retained in ER
- not shipped out
- also known as RxR
- sometimes still accidentally shipped out
- retrieval sequence: KDEL (lys-asp-Glu-leu)
- sent back to ER
- KDEL receptor proteins in golgi
- receptor changes into vesicle to take protein back to ER
- retrograde transport
Golgi resident proteins
- sorted by their own retention/retrieval signals and length of membrane-spanning domains
- not all membrane thickness is the same
- golgi resident proteins will fit into compartment where membrane width matches width of membrane-spanning domain
Lysosomes
- lysosomes = membrane bound organelles important for intracellular digestion
- break down macromolecules via hydrolysis reactions into their smaller monomers so that cell can use monomers
- many types of enzymes in lysosome
- all have optimal activity at an acidic pH (acid hydrolases)
- pump protons to keep pH low
- all must be directed through secretory pathway
- all have optimal activity at an acidic pH (acid hydrolases)
Targeting acid hydrolases to lysosome
- uses sugar tags
- M6P (mannose-6-phosphate)
- same process as quality control… but many manoses
- lysosomal enzymes are glycosylated in ER
- arrives at golgi, one mannose is phosphorylated making M6P tag
- M6P receptor binds to any tag it encounters
- buds off to form vesicle (early endosome)
- low pH in the late endosome/lysosome dissociated the protein from receptor
- receptor returned to golgi
Movement in and out of cell membrane
- exocytosis and endocytosis
- ER and Golgi involved in exocytosis
- electron microscopy tecnique (pulse chase experiment)
Pulse chase experiment
- incubate tissue in radioactive amino acids (ie. Pulse)
- amino acids will get incorporated into newly made proteins
- rinse extra radioactive AAs off
- wait… (ie. chase)
- use radiography film chemicals to cover tissue
- radioactive AA will react and turn black
- analyze location of black spots to figure out where protein located
- do it again to see where proteins go
- use longer chase periods each time
Green fluorescent protein
-more modern to follow protein movement
Sec mutants
- if you dont know how something works, break a part of it and watch to see what happens
- randy shekman (2013)
- Exposed yeast to low levels of mutagens to cause mutations
- Screened yeast to find mutation in secretory pathways (endomembrane system)
- Only picked mutants that were heat sensitive
- normal temps: normal yeast
- high temps: mutated proteins denature
- found many proteins involved in sending glycoproteins through secretory pathways
Exocytosis
- Vesicle moves toward plasma membrane
- Membranes fuse and PM breaks
- Vesicle membrane integrates with PM
- Vesicle contents dumped to outside of cell
- 3 types of exocytosis
- Constitutive: constant flow of material out of cell
- Regulated: vesicle only fuses when receives signal (hormones)
- Polarized: only happens on one side of cell (NTs in neuron)
-when vesicle fuses, proteins in exoplasmic leaflet (facing lumen) remain in that exoplasmic leaflet so then face outside of cell
Endocytosis
- PM invagination, forming pocket of materials
- Pocket begins to pinch off, enclosing materials
- Membrane closes to form vesicle
- Vesicle separates
-vesicle derived from PM, therefore lipids and membrane proteins of PM are brought into cell
Phagocytosis
- example of endocytosis
- engulfment of large particles
- how unicellular cell eats
- in complex organisms involved in immune system
-during phagocytosis vacuole fuses with lysosome and hydrolysis enzymes break down contents
Receptor mediated endocytosis
- extracellular materials bind to receptors on PM in regions called coated pits
- requires clathrin (coat protein), adaptor proteins, and dynamin
- for particular solutes that need to be brought into cell
- Ligand binds to coated pits to form receptor-ligand complexes
- Lateral diffusion across PM
- Clathrin coated pit invaginates
- Dynamin pinches vesicle off to form coated vesicle
Vesicle formation
- coat proteins act as mechanical device that assemble to produce a force which will then curve the membrane until it forms a bubble
- coat proteins very selective about what components will get included in vesicle
-clathrin: coat protein
-COPI: vesicles that leave Golgi
COPII: vesicles that leave ER
Caveolin: cholesterol uptake
-coat proteins always form on cytosolic face
Clathrin coated vesicles
- forms triskelion structures
- made from 3 light chains and 3 heavy chains
- overlap with eachother and cause vesicle to form soccer ball shape
- clathrin also needs adaptor proteins to form connected between membrane receptors and clathrin coat
- AP2 and GGA
AP2
- forms a connector between membrane receptor and clathrin coat
- vesicles coming in from plasma membrane
GGA
- connecter between receptor and clathrin coat
- vesicles leaving golgi to go to lysosomes
- M6P receptor (MPR)
Dynamin
- GTP binding protein
- pinches vesicle off from membrane
- forms ring around stalk of vesicle
- hydrolyzes GTP which causes rings to tighten and squeeze off vesicle
-when GTP cannot be hydrolyzed, vesicle never pinches off… dynamin just keeps being added to be elongated
COPI
- retrograde transport (backwards)
- golgi back to ER
- trans golgi to cis golgi
- coats made of COPI proteins and small GTP binding protein called ARF
- ARF in cystol
- when it binds GTP is inserts into membrane and recruits COPI proteins forcing curvature of membrane
- after vesicle is formed, GTP hydrolyzes and COPI dissociates
COPII
- anterograde transport
- ER to golgi
-coats made of COPII + Sar1 (small GTP binding protein)
- Sar1 in cytosol, binds GTP, inserts into membrane and recruits COPII proteins (Sec13, Sec31, Sec 23, Sec24)
- recruits Sec23+24 first, and then Sec13,31 to complete complex
-after vesicle formed, GTP hydrolyzes and COPII dissociates
COPII coated vesicles summary
- Collect proteins that are destined to leave ER
- ER export signals interact specifically with COPII proteins of the vesicle coats
- Sar1 binds GTP and becomes activated
- Hydrophobic tail swings out of Sar1 and enters lipidbilayer which starts to curve membrane
- Sar1-GTP recruits 4 COPII polypeptides to form coat
- Vesicle buds off
- Sar1 hydrolyze GTP and coat falls off
Summary of coating vesicles
- clatherin coated vesicles:
- trans golgi —>out
- PM —>endosomes
- COPI coated vesicles
- retrograde
- golgi —> ER
- trans golgi —> cis golgi
- retrograde
- COPII coated vesicles
- anterograde
- ER—> golgi
- anterograde
Targeting vesicles to specific compartments
-SNARE hypothesis
- Rabs=small, GTP binding proteins, specify vesicle destination
- 60 types of rabs depending where it has to go
- rabs associate with membrane via a lipid anchor
- rabs recruit tethering proteins to loosely attach the vesicle
- bring SNARES in close proximity
- targeting and tethering
- SNARES= membrane proteins that mediate vesicle fusion
- t-snares = on target membrane (SNAP25 + Syntaxin)
- v-snares on vesicle membrane (synaptobrevin)
- involved in docking
- have alpha helices domains that coil together tightly to bring membranes into close proximity
- due to closeness membranes fuse together
- fusion of membranes is spontaneous
- dissociation of proteins requires energy
NSF
-required to dissociate SNARES
- botulin toxin chops up SNARE proteins
- vesicles cannot fuse
- paralysis caused due to no NT release in NMJ
Autophagy
- destruction of old/sick organelles by isolation in a double membrane vesicle
- followed by fusion with a lysosome
- Yoashinori Ohsumi 2016
- Nobel prize
- organelle is wrapped in double membrane from ER
- called autophagosome once encapsulated
- fuse with lysosome
- broken down and recycled
- some indigestible (residual body)
-residual body build up contribute to aging
