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Co-translational Localization to the Rough ER
5-7Mar2012
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IL-1 is a hormone which lacks a signal sequence from its primary aa sequence.
Devise approach to ascertain mechanism of IL-1 secretion
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Eukaryotic cells compartmentalized with organelles.
- Favorable chemical environment for specialized reactions
- Proteins localized to compartments
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Organelles provide ....
Reduced space/energy requirements for enzymatic reactions
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Translation occurs in the ___, but some proteins are needed in specific ___.
cytoplasm, organelles
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Name three protein sorting mechansims.
- Gated Transport
- Transmembrane transport
- Vesicular Transport
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Gated transport is not a ___.
pore
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In gated transport, proteins move between ___ and ___ thru ___.
- the cytosol
- the nucleus
- nuclear pore complexes
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Transmembrane transport requires that proteins synthesized in the ___ be ___ in the ___.
cytoplasm, refolded, organelle
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In transmembrane transport, ___ directly transport specific proteins ___.
- transmembrane protein translocators
- across a membrane from the cytosol into a space which is topologically distinct
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Vesicular transport occurs ___
From the rough ER to Golgi to membrane/lysozome
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In vesicular transport, ___ ferry proteins from one compartment to another and deliver cargo by ___.
- vesicles
- fusing with the membrane enclosing the destination compartment
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Describe Free Ribosome Cycle
- mRNA encoding cytosolic protein remains free in cytosol
- Multiple copies of protein can be made with free polyribosome in the cytosol
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Describe the Membrane-Bound Ribosome Cycle
- mRNA starts translation of protein
- ER signal sequence is translated
- Signal Recognition Particle (SRP) binds to signal sequence
- Bound SRP/ribosome complex is then bound to ER where translation continues
- Polyribosome bound to ER membrane by multiple nascent polypeptide chains
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Proteins translated by free ribosomes ___, ___, and ___.
- stay in the cytosol
- are not glycosylated
- do not have disulfide bonds
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Proteins translated on Rough ER are ___/___.
modified/glycosylated
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The enzymes for disulfide bonds and ___ are only in the ___.
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What is the benefit of disulfide bonds?
More stable structure (e.g. hormones and antibodies)
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Proteins are sorted to organelles according to ___ found in their ___.
- signal sequences
- aa sequence
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Terminal end signal sequences are ____ after sorting while ___ are not.
- removed
- internal ones forming a signal patch
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Signal sequences are both ___ and ___ for protein targeting.
Necessary, sufficent
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___ properties of signal sequences such as ___ seem more important than the exact aa sequence.
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Signal sequences are recognized by ___ that ___. They function ___, i.e. return to point of origin to be reused.
- complementary sorting receptors
- guide proteins to their appropriate destination
- catalytically
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Describe a signal patch.
- Made of several regions which come together when folded.
- Not removed after sorting.
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Why aren't signal patches removed after sorting?
- During chloroplast and mitochondrial evolution, they were separate bacteria
- No mechanism for removal at that time.
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Why does the signal sequence for the ER have such a long sequence with a significant hydrophobic region?
The ER has transmembrane proteins unlike other organelles.
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What are some techniques for studying protein sorting?
- Transfection of recombinant proteins with localization signals
- Protein translocation, cell-free assays
- Genetic approach
- Microscopy
- Ultracentrifugation of vessicles
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Describe transfection approach.
- Clone localization signal for an organelle.
- Recombine using plasmid with gene normally found in cytoplasm and GFP
- Inject into cell and see where protein ends up
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Describe biochemical technique for studying protein translocation.
- Protein labeled with radioactive signal sequence
- Analytic/rate zonal centrifugation
- Two fractions - solo and internal to organelle
- Organelle protease removes signal sequence
- SDS-PAGE shows less massive protein
- Protein protected in organelle when protease added to medium
- Protein susceptible if detergent also added
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What protein translocation technique is better than centrifugation?
Antibody with immunofluorescence
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Describe genetic technique for studying translocation.
- Engineered cell dies as enzyme is translocated to ER at permissive temp
- Mutant cell lives since translocation apparatus is broken
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Describe microscopy and translocation
- Nuclear import fails due to mutation of single a.a.
- Phenotype visible with microscope
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Describe Cell-free assay technique
- Rate zonal centrifugation
- Smooth microsomes - low density
- rough microsomes - high density
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What are the two types of protein translocation?
- Co-translation
- Post-translation
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Which type of translocation is for the rough ER?
Co-translation
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What does post-translation cover?
Mitochondria, chloroplasts, nucleus, peroxisomes
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The signal sequence is recognized by ___.
the signal recognition particle
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What is an SRP composed of?
protein and RNA
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What cleaves the signal sequence?
A signal peptidase
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When does folding of a protein occur?
At end of translation
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Will proteins fold by themselves? In all cases?
- No, they need assistance.
- Tiny ones may fold by themselves.
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Describe the SRP's mechanism of action.
- SRP binds to signal peptide
- Translation is stalled
- Ribosome/protein complex binds to rough ER
- Translation continues
- Protein is translocated into the rough ER lumen
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Why is the small RNA needed in the SRP?
Interacts with ribosome to stall translation
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Describe the SRP's structure.
- 6 proteins and 1 RNA
- Translational pause domain
- RNA molecule
- Hinge
- GTPase and SRP receptor binding site
- Signal-sequence binding pocket
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Describe a translocator
- In ER membrane
- Gated pore
- Signal sequence opens gate/removes plug when docked
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What are three ways protein translocation can be driven thru structurally similar translocators?
- Co-trans - Co-translational - ribosome/protein complex to ER and interacts with Sec61
- post (euk) - sec62,63,71,72 complex; ATP/BiP-driven
- post (bac) - SecA/ATP-driven
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What translocation elements are found in all organisms?
- Sec61 (translocator)
- SRP
- SRP receptor
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Describe types of co-translational transport.
- Two-signal - start transfer sequence (cleaved) and stop transfer sequence.
- Internal start transfer sequence - not cleaved.
- Two-signal - neither cleaved
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What part of the translocated protein gets glycoslyated?
The part in the ER
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For protein with an internal signal sequence, which side ends up in the cytosol?
The more positively-charged side
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For a translocated protein, which side will be located outside of the cell?
The side in the ER
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If the hydrophobic end of a translocated protein is exposed to the cytoplasm, what happens?
The hydrophobic end will try to bind to other cells
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What are two types of glycosylation?
- 90% N-linked: asparagine-X-Ser; catalyzed by membrane-bound protein; rough ER
- O-linked: Hydroxy oxygen of a.a.'s; Golgi
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Why is glycosylation done before translation is finished?
Lipid-linked saccharides can affect folding. Proteins leave the ER only when properly folded.
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Intracellular Protein Sorting
7-12Mar2012
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In general, what happens to proteins that aren't folded?
They are removed.
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What are three possible mechanisms for ER-Golgi transport?
- Vesicles/tubules
- Direct connection
- Transient connections
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Describe vesicular transport
- COPII-coated
- Secretion of small cargo (60-90nm)
- Vesicles moved to Golgi on microtubles
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Describe tubular transport
- Transport of large molecules
- From ER to Golgi
- Between Golgi cisternae
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Describe a class 1 transport mutant.
- COPII temp-sensitive mutants resulted in accumlation of cargo in the ER.
- Prevents formation of vesicles
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Describe a class 2 transport mutant.
- Unable to dock on the Golgi membrane.
- Proteins used for docking on golgi and/or vesicles defective.
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Describe ER export sites (ERES).
- aka transitional ER.
- Involved in export and selection of cargo.
- Export sequence motifs required for selection and transport.
- COPII proteins present.
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What are the two coat protein complexes involved in ER-Golgi transport?
COPI and COPII
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Describe COPI
- Involved in retrograde movement of vesicles.
- i.e. Golgi to ER for recycling of components
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Describe COPII
- ER to Golgi
- Interact with SNARES to form vesicles
- Recognizes HDEL(yeast)/KDEL(mammals) motifs for export
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The Golgi complex is ___. Different proteins go to ___ parts of the Golgi.
- compartmentalized
- different
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Why is cargo in ER/Golgi selected for?
- Direct vesicles to correct location affecting chemical modification.
- Groups of vesicles may have final destination in common.
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Describe coat formation.
- Similar to clathrin-coated vesicles
- G-proteins required for membrane recruitment and curvature
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COP1 and COPII coat formation leads to ___.
- Cargo concentration
- Membrane curvature
- Vesicle budding
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When a vesicle is formed, ___ recruits ___ to form a coat.
Sec12p, Sar1p (GTPase)
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___ removes the protein coat.
GTP hydrolysis
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Rapid GTP hydrolysis leads to ___ while slower hydrolysis leads to ___.
- formation of tubles
- formation of vesicles
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The rate of hydrolysis is also affected by ___.
Size of cargo
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Protein cargo interacts with ___. Therefore, it is involved in ___.
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How many protein motifs are involved in cargo and ___ interaction?
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___ and other proteins act as ___ that ___ in the lumen.
SNARES, receptors (docking), select cargo
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ER->Golgi transport is ___ -driven.
dynein (motor) / dynactin (bind)
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What is a molecular method to show dynactin is required?
- Knockdown gene for dynactin
- Timelapse w/GFP and compare to WT
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How would you label microtubule to see dynactin and cargo?
- Label dynactin with antibody w/GFP
- Label tubulin with red
- Overlap will appear yellow
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How are vesicles recognized by the docking membrane?
Rabs (GTPase) and SNARES
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Describe two types of SNARES.
- v-SNARES on vesicles
- t-SNARES - on target membrane
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SNARES are ___ to a protein.
specific
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SNARES must ___ in order to be used again for ___.
dissociate, docking
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___ dissociates SNARES.
NSF powered by ATP
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Adapter proteins on the NSF are recycled by vesicles with ___ back to the ___.
COPI, Rough ER
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What is the advantage of vesicles binding together?
Can accumulate large amounts of cargo
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Compare different coated vessels
- Clathrin - from plasma membrane and TGN
- COPI/II - between Golgi cisternae; between ER and Golgi
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Describe vesicle transportation between ER and Golgi
- Vesicle buds with Rab-GTP and v-SNARE
- Transports across
- Rab effector binds with Rab-GTP for docking and providing specificity
- t-SNARE and v-SNARE bind allowing for membrane fusion
- Membrane fusion occurs
- GTP is hydrolyzed
- Rab-GDP get recycled back to Rough ER binding to GEF
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If vesicles are released from ER but don't dock to Golgi, what might be wrong?
- Rab might be defective
- t/v-SNARES might be defective
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How would you trouble-shoot docking problem with vesicle to Golgi?
- Introduce mRNA for rescue of v/t-snare, Rab protein, Rab effector, GEF
- Or PCR looking for mutations and then do functional assays
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Transport from the Golgi
12Mar2012
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What part of the golgi is responsible for glycolsylation?
The medial stack
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In the Golgi cell-free assay, why are cytosol and ATP added?
- cytosol - essential proteins (motor, binding); microtubules
- ATP - energy for motor proteins
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In the Golgi cell-free assay, how do you measure incorporation of sugar group?
Tag with radioactivity (for example)
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Describe the Trans-Golgi Network (TGN).
- Distal surface of trans side of Golgi.
- Export to cell membrane and endosomes/lysosomes.
- Recognition and selection of TGN export sites.
- Clathrin-coated vesicles used for transport.
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When is a clathrin coat used?
- TGN to cell membrane or lysosome.
- Endocytosis vesicles that form at membrane and go to lysosome.
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What is needed to target proteins to the lysosome?
Mannose-6-phosphate
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What are the three best-understood pathways of protein sorting from the TGN?
- Signal-mediated to lysosomes.
- Signal-mediated to secretory vesicles (e.g. insulin).
- Constitutive secretory pathway (e.g. collagen).
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Describe clathrin coat structure
- Polyhedral composed of 36 triskelions.
- 3 heavy chains
- 3 light chains
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Describe the assembly and disassembly of the clathrin coat.
- Bind to adaptin which binds to cargo.
- Bud formation occurs.
- Vesicle forms and is cut away by dynamin/proteins.
- Coat sloughs off leaving naked transport vesicle.
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How is pH regulated between the ER, vesicular tubular cluster, and Golgi?
Ion transporters in the membrane
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Where does glycosylation begin?
In the rough ER
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List the parts of the Golgi from closest to farthest to ER.
- cis Golgi network.
- cis cisterna
- medial cisterna
- trans cisterna
- trans Golgi network
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What function occurs at the cis Golgi network?
- Sorting.
- Phosphorylation of oligosaccharides on lysosomal proteins (e.g. M6P)
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What function occurs at the cis cisterna?
Removal of Man (not mannose-6-phosphate)
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What function occurs at the medial cisterna?
- Removal of Man
- Addition of GlcNAc
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What function occurs at the trans cisterna?
- Addition of Gal
- Addition of NANA
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What function occurs at the trans golgi network?
- Sulfation of tyrosines and carbs.
- Sorting to lysosome, plasma membrane, secretory vesicles.
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What's the difference between phago and endo cytosis?
- Phago - large (e.g. cells)
- Endo - small items (often in large volumes)
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How are lysosomal proteins recognized in the TGN?
Mannose-6-phosphate (M6P) groups recognized by receptors.
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Name three pathways to the lysosome.
- Phagocytosis -> phagosome - not clathrin-coated.
- Endocytosis -> endosome - clathrin-coated shed after budding.
- Autophagy -> autophagosome
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Describe the transport of lysosomal hydrolases to lysosomes.
- Precursor comes from ER with mannose.
- Phosphate added in cis golgi network.
- Binding, budding and clathrin coat at TGN.
- Transport via bound M6P receptor.
- Fusion/docking (SNARES) to late endosome.
- Dissociation from vesicle/receptor at higher pH.
- Removal of phosphate.
- Lysosomal hydrolase now mature/active.
- Receptor/vesicle recycled back to TGN.
- Late endosome fuses with lysosome.
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What does the phosphate do on the lysosomal hydrolase.
It keeps the enzyme inactive.
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How is the lysosomal hydrolase recognized in the Golgi?
A signal patch
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Specialized secretory cells have a ___ secretory pathway.
regulated
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Selected proteins in the TGN are diverted into ___, where they are stored until ___.
- secretory vesicles.
- an extracellular signal stimulates their secretion.
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Some secreted small molecules are often ___ so that they can be stored ___.
- complexed to specific macromolecules
- at high conc w/o generating a high osmotic pressure
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How are secretory vesicles formed?
- They aggregate in ionic environment of TGN.
- Often become condensed and lumen becomes more acidic.
- New vesicles form picking up clathrin patches and membrane.
- Mature secretory vesicle forms shedding clathrin.
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Describe use of temp sensitive mutations in rough ER.
- Induce random mutations.
- Select phenotypes that fail to translocate enzyme into rough ER.
- At restrictive temp, transporter is not folded correctly.
- Enzyme stays in cytoplasm.
- Histidine not needed in culture.
- PCR SRP and translocator
- Do functional assay/rescue to verify
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Describe a type 1 chaperonin.
- Bacterial/mitochondia/chloroplast
- 14mer
- Separate lid
- post-translational
- 60 kDa
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Describe a type 2 chaperonin.
- Archaea and eukarya
- 8mer
- Build-in lid
- mostly cotranslational
- 50 kDa
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