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Co-translational Localization to the Rough ER
IL-1 is a hormone which lacks a signal sequence from its primary aa sequence.
Devise approach to ascertain mechanism of IL-1 secretion
Eukaryotic cells compartmentalized with organelles.
- Favorable chemical environment for specialized reactions
- Proteins localized to compartments
Organelles provide ....
Reduced space/energy requirements for enzymatic reactions
Translation occurs in the ___, but some proteins are needed in specific ___.
Name three protein sorting mechansims.
- Gated Transport
- Transmembrane transport
- Vesicular Transport
Gated transport is not a ___.
In gated transport, proteins move between ___ and ___ thru ___.
- the cytosol
- the nucleus
- nuclear pore complexes
Transmembrane transport requires that proteins synthesized in the ___ be ___ in the ___.
cytoplasm, refolded, organelle
In transmembrane transport, ___ directly transport specific proteins ___.
- transmembrane protein translocators
- across a membrane from the cytosol into a space which is topologically distinct
Vesicular transport occurs ___
From the rough ER to Golgi to membrane/lysozome
In vesicular transport, ___ ferry proteins from one compartment to another and deliver cargo by ___.
- fusing with the membrane enclosing the destination compartment
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
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
Proteins translated by free ribosomes ___, ___, and ___.
- stay in the cytosol
- are not glycosylated
- do not have disulfide bonds
Proteins translated on Rough ER are ___/___.
The enzymes for disulfide bonds and ___ are only in the ___.
What is the benefit of disulfide bonds?
More stable structure (e.g. hormones and antibodies)
Proteins are sorted to organelles according to ___ found in their ___.
- signal sequences
- aa sequence
Terminal end signal sequences are ____ after sorting while ___ are not.
- internal ones forming a signal patch
Signal sequences are both ___ and ___ for protein targeting.
___ properties of signal sequences such as ___ seem more important than the exact aa sequence.
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
Describe a signal patch.
- Made of several regions which come together when folded.
- Not removed after sorting.
Why aren't signal patches removed after sorting?
- During chloroplast and mitochondrial evolution, they were separate bacteria
- No mechanism for removal at that time.
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.
What are some techniques for studying protein sorting?
- Transfection of recombinant proteins with localization signals
- Protein translocation, cell-free assays
- Genetic approach
- Ultracentrifugation of vessicles
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
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
What protein translocation technique is better than centrifugation?
Antibody with immunofluorescence
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
Describe microscopy and translocation
- Nuclear import fails due to mutation of single a.a.
- Phenotype visible with microscope
Describe Cell-free assay technique
- Rate zonal centrifugation
- Smooth microsomes - low density
- rough microsomes - high density
What are the two types of protein translocation?
Which type of translocation is for the rough ER?
What does post-translation cover?
Mitochondria, chloroplasts, nucleus, peroxisomes
The signal sequence is recognized by ___.
the signal recognition particle
What is an SRP composed of?
protein and RNA
What cleaves the signal sequence?
A signal peptidase
When does folding of a protein occur?
At end of translation
Will proteins fold by themselves? In all cases?
- No, they need assistance.
- Tiny ones may fold by themselves.
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
Why is the small RNA needed in the SRP?
Interacts with ribosome to stall translation
Describe the SRP's structure.
- 6 proteins and 1 RNA
- Translational pause domain
- RNA molecule
- GTPase and SRP receptor binding site
- Signal-sequence binding pocket
Describe a translocator
- In ER membrane
- Gated pore
- Signal sequence opens gate/removes plug when docked
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
What translocation elements are found in all organisms?
- Sec61 (translocator)
- SRP receptor
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
What part of the translocated protein gets glycoslyated?
The part in the ER
For protein with an internal signal sequence, which side ends up in the cytosol?
The more positively-charged side
For a translocated protein, which side will be located outside of the cell?
The side in the ER
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
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
Why is glycosylation done before translation is finished?
Lipid-linked saccharides can affect folding. Proteins leave the ER only when properly folded.
Intracellular Protein Sorting
In general, what happens to proteins that aren't folded?
They are removed.
What are three possible mechanisms for ER-Golgi transport?
- Direct connection
- Transient connections
Describe vesicular transport
- Secretion of small cargo (60-90nm)
- Vesicles moved to Golgi on microtubles
Describe tubular transport
- Transport of large molecules
- From ER to Golgi
- Between Golgi cisternae
Describe a class 1 transport mutant.
- COPII temp-sensitive mutants resulted in accumlation of cargo in the ER.
- Prevents formation of vesicles
Describe a class 2 transport mutant.
- Unable to dock on the Golgi membrane.
- Proteins used for docking on golgi and/or vesicles defective.
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.
What are the two coat protein complexes involved in ER-Golgi transport?
COPI and COPII
- Involved in retrograde movement of vesicles.
- i.e. Golgi to ER for recycling of components
- ER to Golgi
- Interact with SNARES to form vesicles
- Recognizes HDEL(yeast)/KDEL(mammals) motifs for export
The Golgi complex is ___. Different proteins go to ___ parts of the Golgi.
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.
Describe coat formation.
- Similar to clathrin-coated vesicles
- G-proteins required for membrane recruitment and curvature
COP1 and COPII coat formation leads to ___.
- Cargo concentration
- Membrane curvature
- Vesicle budding
When a vesicle is formed, ___ recruits ___ to form a coat.
Sec12p, Sar1p (GTPase)
___ removes the protein coat.
Rapid GTP hydrolysis leads to ___ while slower hydrolysis leads to ___.
- formation of tubles
- formation of vesicles
The rate of hydrolysis is also affected by ___.
Size of cargo
Protein cargo interacts with ___. Therefore, it is involved in ___.
How many protein motifs are involved in cargo and ___ interaction?
___ and other proteins act as ___ that ___ in the lumen.
SNARES, receptors (docking), select cargo
ER->Golgi transport is ___ -driven.
dynein (motor) / dynactin (bind)
What is a molecular method to show dynactin is required?
- Knockdown gene for dynactin
- Timelapse w/GFP and compare to WT
How would you label microtubule to see dynactin and cargo?
- Label dynactin with antibody w/GFP
- Label tubulin with red
- Overlap will appear yellow
How are vesicles recognized by the docking membrane?
Rabs (GTPase) and SNARES
Describe two types of SNARES.
- v-SNARES on vesicles
- t-SNARES - on target membrane
SNARES are ___ to a protein.
SNARES must ___ in order to be used again for ___.
___ dissociates SNARES.
NSF powered by ATP
Adapter proteins on the NSF are recycled by vesicles with ___ back to the ___.
COPI, Rough ER
What is the advantage of vesicles binding together?
Can accumulate large amounts of cargo
Compare different coated vessels
- Clathrin - from plasma membrane and TGN
- COPI/II - between Golgi cisternae; between ER and Golgi
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
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
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
Transport from the Golgi
What part of the golgi is responsible for glycolsylation?
The medial stack
In the Golgi cell-free assay, why are cytosol and ATP added?
- cytosol - essential proteins (motor, binding); microtubules
- ATP - energy for motor proteins
In the Golgi cell-free assay, how do you measure incorporation of sugar group?
Tag with radioactivity (for example)
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.
When is a clathrin coat used?
- TGN to cell membrane or lysosome.
- Endocytosis vesicles that form at membrane and go to lysosome.
What is needed to target proteins to the lysosome?
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).
Describe clathrin coat structure
- Polyhedral composed of 36 triskelions.
- 3 heavy chains
- 3 light chains
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.
How is pH regulated between the ER, vesicular tubular cluster, and Golgi?
Ion transporters in the membrane
Where does glycosylation begin?
In the rough ER
List the parts of the Golgi from closest to farthest to ER.
- cis Golgi network.
- cis cisterna
- medial cisterna
- trans cisterna
- trans Golgi network
What function occurs at the cis Golgi network?
- Phosphorylation of oligosaccharides on lysosomal proteins (e.g. M6P)
What function occurs at the cis cisterna?
Removal of Man (not mannose-6-phosphate)
What function occurs at the medial cisterna?
- Removal of Man
- Addition of GlcNAc
What function occurs at the trans cisterna?
- Addition of Gal
- Addition of NANA
What function occurs at the trans golgi network?
- Sulfation of tyrosines and carbs.
- Sorting to lysosome, plasma membrane, secretory vesicles.
What's the difference between phago and endo cytosis?
- Phago - large (e.g. cells)
- Endo - small items (often in large volumes)
How are lysosomal proteins recognized in the TGN?
Mannose-6-phosphate (M6P) groups recognized by receptors.
Name three pathways to the lysosome.
- Phagocytosis -> phagosome - not clathrin-coated.
- Endocytosis -> endosome - clathrin-coated shed after budding.
- Autophagy -> autophagosome
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.
What does the phosphate do on the lysosomal hydrolase.
It keeps the enzyme inactive.
How is the lysosomal hydrolase recognized in the Golgi?
A signal patch
Specialized secretory cells have a ___ secretory pathway.
Selected proteins in the TGN are diverted into ___, where they are stored until ___.
- secretory vesicles.
- an extracellular signal stimulates their secretion.
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
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.
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
Describe a type 1 chaperonin.
- Separate lid
- 60 kDa
Describe a type 2 chaperonin.
- Archaea and eukarya
- Build-in lid
- mostly cotranslational
- 50 kDa
What is aPKC?
atypical protein kinase which controls cell polarity in many types of cells.
What is Baz/Par-3?
Forms Par/aPKC complex controlling cell polarity.
What is Par/aPKC complex?
- Localizes to apical cortex.
- Responsible for basal localization of cell determinants.
What is Miranda (Mira)?
- Cell fate determinant affected by aPKC/Par complex.
- In NB - basal cresent.
- Not epithelial.
What is exo84[onr]?
- hypomorphic allele of exo84
- mutants display epithelial defects resulting in mislocalization of apical junction proteins including baz.
What is shibire?
Temperature sensitive allele of dynamin.
- Testing loss of exocyst function.
- Homozygous exo84[onr] has no effect on NB baz or mira.
- Homozygous exo84[onr] mislocalizes baz in epithelial.
- Testing loss of dynamin function using temp-sensitive allele.
- Severe disruption of epithelial organization.
- No effect on NB baz nor mira on larva and embryo.
Testing regulators of vesicle trafficking using mosaics in brain.