Week 5 Flashcards
Cytosol
The regions inside the plasma membrane but outside all of the membrane bound organelles.
Cytoplasm
The area outside of the nucleus and inside the plasma membrane
How much of the total membrane in a eukaryotic cell is found in the plasma membrane?
Only about 2-5%.
Signal hypothesis
Proteins that leave the cytosol have intrinsic signals that direct them to the appropriate organelle.
Cis-acting signals
Signals within the protein that provide its address label.
A sorting signal can be found in which level of structure of a protein?
A sorting signal can be part of the primary, secondary, tertiary, or quaternary structure of a protein, or a posttranslational modification.
The protein transport pathway from the cytosol to the nucleus is an example of what type of transport?
Gated transport.
Protein transport from the cytosol to the mitochondria, ER, and plastids is an example of what type of transport?
Transmembrane transport.
Gated transport
Fully folded proteins pass through a large pore (nuclear pore).
Translocation
Unfolded proteins are threaded through a small pore (ER translocon). Membrane remains impermeable to small molecules.
Where does translocation occur?
In protein transport to the ER, mitochondria, and chloroplasts.
Vesicular transport
Proteins move between organelles without crossing a membrane. Carried by small membrane-bound vesicles.
In what type of pathways does vesicular transport take place?
Secretory and endocytic pathways.
NLS
Nuclear localization signal
How was NLS discovered?
Using molecular biology approaches such as necessary and sufficient tests.
How is NLS related to position on the polypeptide?
The NLS is position independent and is never cleaved off of the protein after it is used.
Molecular biology approach for transplanting a potential signal sequence onto a reporter protein
The DNA sequence coding for the signal is fused in frame with the gene coding for the reporter. The fusion gene is transfected into cells and the fusion protein is expressed.
Sufficiency test for the T-antigen NLS
Transplant the putative localization signal onto a cytosolic protein. Ask: Is the signal sufficient to localize the fusion protein to the nucleus?
How is the signal for a sufficiency test for the T-antigen NLS detected?
T-antigen NLS is fused to pyruvate kinase (cytosolic enzyme). It can then be detected by indirect immunofluorescence using anti-pyruvate kinase antibodies.
Necessary test for the T-antigen NLS
Mutate the putative signal in the context of the full length protein. Ask: Is the signal necessary to localize the protein to the nucleus?
How are the results of the necessary test for the T-antigen NLS evaluated?
Each bright structure is a nucleus containing wild-type T-antigen. The mutant T-antigen localizes to the cytosol. The nucleus is the dark region of the cells.
How does T-antigen, or any other nuclear protein, get through the nuclear envelope?
The nuclear pore.
Nucleoporins
Individual proteins in the nuclear pore
How many unique proteins make up the nuclear pore?
~30
Size of the nuclear pore
The nuclear pore is a 60 MDa complex
Size of molecules that enter the nucleus by free diffusion
< 40 kDa
Size of macromolecules that enter the nucleus by active transport
> 40 kDa
Rate of transport into the nucleus through the nuclear pore
~ 100 histone proteins/pore/minute
Oily sphaghetti model
Model of the nuclear pore that says “greasy” loops of protein extend into the nuclear pore and act as a barrier.
The “postmen” required to deliver cargo into the nucleus
Nuclear import receptors and Ran
Ran
Small monomeric GTP binding protein
Ran GAP
GTPase activating protein
Ran GEF
Guanine nucleotide exchange factors.
Role of Ran GEF
Kicks out GDP and allows GTP binding
Importins
Cargo receptors for nuclear proteins
Role of Ran
Ran regulates importin in the process of cargo dissociation.
Localization of Ran-GTP
High in the nucleus and low in the cytosol
What causes the binding and dissociation of cargo in nuclear transport?
Mutually exclusive binding of Ran-GTP and cargo to the import receptor. Therefore, Ran-GTP binding causes dissociation of cargo
What causes the binding of cargo in nuclear export?
Mutually dependent binding of Ran-GTP and cargo to the export receptor. Therefore, Ran-GTP binding causes association of cargo.
What is the role of T-cells, and how are they activated?
T-cells help mediate immune responses and are activated by a calcium influx into the cytosol.
Effect of calcium influx into the cytosol.
Calcium activates a calcium-dependent protein phosphatase called calcineurin, which dephosphorylates NF-AT.
NF-AT
A transcription factor regulating T-cell response.
Effect of immunosuppressive drugs such as cyclosporin A and FK506
These drugs inhibit dephosphorylation of NF-AT.
hnRNPs
Heterogenous ribonucleoprotein particles
Role of hnRNPs
As RNA is transcribed, it is packaged with hnRNPs. Some of the hnRNP proteins stay in the nucleus while others shuttle back and forth. Nuclear export signal in hnRNP A1 contributes to mRNA export from the nucleus.
Nucleolus
The region of the nucleus where ribosomes are assembled.
What are ribosomes formed from?
Ribosomes are formed from a loop of DNA (chromosome) that carries multiple tandem copies of rRNA genes.
Composition of the 40S subunit.
The 40S subunit has 33 different proteins and one rRNA molecule.
Composition of the 60S subunit
The 60S has 46 proteins and 3 rRNAs.
Where are ribosomal subunits assembled?
The subunits are assembled in the nucleus and exported to the cytosol.
What type of reactions occur in the peroxisomes of mammals?
- Oxidative and peroxidative reactions (detox).
- RH2 + O2 –> R + H2O2
- Catalase converts 2 H2O2 –> 2 H2O and O2
- B-oxidation of lipids, particularly very long chain fatty acids (breakdown to 2-carbon units)
Role of peroxisomes for plants
- Photorespiration in leaves
- Oxidation of lipids (breakdown of acyl chains 2 carbons at a time) for biosynthetic use
- Glyoxylic acid cycle in plants allows conversion of fats to sugars.
Before GFP was discovered, what was firefly luciferase used for?
It was developed for use as a reporter in gene fusion experiments.
General reaction for using luciferase to make light
Luciferase + luciferin + ATP -> light
How was luciferase used to discover a peroxisomal targeting signal?
- Firefly luciferase localized to peroxisomes when expressed in mammalian cells, plants, or yeast (co-localizes with catalase)
- Necessary and sufficient tests determined that the C-terminal SKL is luciferase’s peroxisomal targeting signal (PTS)
Pichia pastoris
Methanol-eating yeast
Results when Pichia are grown on methanol as the sole carbon source
They are filled with peroxisomes and express high levels of methanol oxidase, a peroxisomal protein.
What is required for Pichia to grow on either methanol or fatty acids (oleic acid) as the sole carbon source?
Peroxisomes
What technique was performed to identify mutant yeast that could not grow on either methanol or oleic acid as the sole carbon source?
Genetic screens
Result of the genetic screens on mutant yeast
These screens identified the pex (peroxin) mutants, many of which exhibited peroxisome “ghosts” by electron microscopy.
How were the PEX genes identified in yeast analyzed?
They were cloned by complementation and the proteins were characterized.
What was the rationale/hypothesis for the Pichia pastoris experiments?
Because the biochemical pathways for methanol and fatty acid breakdown are very different, the assumption was that a single gene mutation that disrupted both of these pathways would likely disrupt peroxisome biogenesis (the one thing both pathways have in common is that they occur within peroxisomes.)
What were the four mediums that Pichia were grown on?
- Glucose
- Methanol
- Oleic acid
- Ethanol
PTS1
The C-terminal SKL sequence
PTS1R
The receptor that binds the SKL sequence (AKA Pex5)
Describe the model for peroxisomal protein import.
The receptor (PTS1R or Pex5p) brings cargo to the peroxisomal membrane where other Pex proteins form a translocation pore and mediate the release and recycling of the receptor. Fully folded catalase can cross the peroxisome membrane.
Zellweger Syndrome
A type of peroxisome biogenesis disorder (PBD). Physical presentation: High forehead, epicanthal folds, and hypoplasia of supraorbital ridges and midface.
Involvement of fibroblasts in PBDs
Fibroblasts from many patients are deficient for uptake of peroxisomal proteins.
Describe the mitochondria membranes.
Mitochondria have an inner and outer membrane that defines two distinct compartments, the intermembrane space and the matrix
Role of mitochondria and how they accomplish it
ATP generation. Electron transport in mitochondria and photosynthesis in chloroplasts. Also involved in lipid synthesis
Describe the chloroplast membranes.
Chloroplasts have three membranes: outer, inner, and thylakoid that define the intermembrane space, the stroma, and thylakoid lumen.
What is the mitochondrial protein signal sequence?
An N-terminal alpha-helix with basic residues on one face. The secondary structure is important for this signal.
Role of TIMs and TOMS
TIMs and TOMs form the translocation channel and signal sequence receptors for mitochondrial protein import.
TOM translocation
Across the outer membrane
TIM translocation
Across the inner membrane
Upon arrival in the mitochondrial matrix, what often happens to the signal sequence?
The sequence is cleaved by signal peptidase.
What are the energy requirements for mitochondrial protein import?
Hsp70: Heat shock protein of 70 kDa. ATPase needed to maintain the unfolded state of the precursor in the cytosol. A mitochondrial Hsp70 is also required in the matrix to facilitate unidirectional translocation. The electrochemical proton gradient, generated by the electron transport chain, is also required to drive the matrix protein across the inner membrane.
What encodes mitochondria and chloroplast proteins?
Mitochondria and chloroplasts have their one genome, but the majority of their proteins are encoded by nuclear genes.
Where are nearly all proteins that are secreted synthesized?
Nearly all proteins that are secreted, or are part of the nuclear envelope, ER, Golgi, secretory vesicles, plasma membrane, endosomes, and lysosomes are initially synthesized at the ER.
Where is most of the phospholipid and sterol for the entire cell synthesized?
The ER. The ER is a biogenic membrane.
What is the lumen of the nuclear envelope, ER, Golgi, secretory vesicles, endosomes, and lysosomes topologically equivalent to?
The outside of the cell.
Which organelles compose the secretory pathway?
The ER, Golgi, and secretory vesicles.
Which organelles compose the endocytic pathway?
The endocytic vesicles, endosomes, and lysosomes.
What pulls the ER out of the nuclear envelope?
The ER is pulled out of the nuclear envelope along microtubule tracks by a plus end directed motor protein (a kinesin).
MTOC
Microtubule organizing center
What concentrates the Golgi at the MTOC?
The Golgi is concentrated at the MTOC by a minus end motor.
When calcium is high in the cell, what does it bind to? What effect does this have?
Calcineurin. This initiates the regulation of nuclear transport.
Nucleolus
A protein-rich region where ribosomes are produced. Stains darkly on TEM micrograph.
What happens to the ER in a cell lysate?
The ER fragments into microsomes in a cell lysate.
Role of smooth ER
Produces most of the lipids (sterols and phospholipids) for the cell. Detox of hydrophobic compounds.
What determines the density of an organelle?
The protein to lipid ratio. The higher the protein content, the higher the density.
