1 Flashcards
(14 cards)
Identify the components and steps to reconstitute protein import in a test tube with purified ER or mitochondria
• For ER: You would use purified ER microsomes, ribosomes, mRNA coding for the target protein, and necessary cytosolic factors like SRP. Adding ATP and GTP allows co-translational translocation to occur.
• For mitochondria: Purified mitochondria, precursor proteins, cytosolic extract (with Hsp70), ATP, and a membrane potential are required for protein import.
Describe how removal of signal sequences or MTSs after import impacts the way a protein runs on an SDS-PAGE gel
Removal of signal sequences or MTSs after import will result in a shift in molecular weight. On an SDS-PAGE gel, the protein will migrate faster because the signal or targeting sequence, which adds mass, has been cleaved. This shift in migration can be used to confirm successful import into the ER or mitochondria.
Describe what a protease protection assay is and how it can be used to monitor import into an organelle in combination with western blotting
A protease protection assay is used to determine whether a protein has been successfully imported into an organelle.
After incubation with the organelle, protease is added. Proteins inside the organelle are protected from degradation, while exposed proteins are degraded. A western blot can then be used to detect which proteins remain intact.
Describe what N-linked glycosylation is and how it can be used to monitor import into the ER
N-linked glycosylation is the attachment of an oligosaccharide to the nitrogen atom of an asparagine residue on a protein. It occurs in the ER and helps monitor protein import. If a protein is glycosylated, it means it has successfully entered the ER lumen, as this process only occurs there. It can be detected using methods like western blotting to confirm successful import.
Describe what N-linked glycosylation is and how it can be used to monitor import into the ER
N-linked glycosylation is the attachment of an oligosaccharide to the nitrogen atom of an asparagine residue on a protein. It occurs in the ER and helps monitor protein import. If a protein is glycosylated, it means it has successfully entered the ER lumen, as this process only occurs there. It can be detected using methods like western blotting to confirm successful import.
• Outline steps involved in importing proteins into each of the different compartments of the mitochondria (outer membrane, inner membrane, inner membrane space, matrix), identify the protein components of the machinery involved, and what types of energetic forces are required (ATP, membrane potential, etc.) at each step
Steps for importing proteins into the mitochondria
Mitochondrial proteins are synthesized in the cytosol and imported post-translationally. There are four major pathways depending on the destination.
- Outer membrane:
o TOM complex: Translocates proteins across the outer membrane.
o Some outer membrane proteins are then inserted into the membrane by the SAM complex. - Intermembrane space:
o After passing through the TOM complex, proteins destined for the intermembrane space have their intermembrane space targeting sequence cleaved. - Inner membrane:
o Proteins pass through the TIM23 or TIM22 complex, depending on their structure.
o Membrane potential across the inner membrane drives the insertion of proteins via TIM complexes. - Matrix:
o Proteins with an N-terminal matrix targeting sequence are translocated by the TOM and TIM23 complexes.
o ATP hydrolysis and the mitochondrial Hsp70 chaperone pull the protein into the matrix.
Outline the steps involved in importing proteins into the ER, including machinery involved at each step and how the process differs for soluble and membrane proteins
Steps for importing proteins into the ER
• Targeting to the ER: Proteins with an N-terminal signal sequence are recognized by the signal recognition particle (SRP) as they are being synthesized.
• SRP binding: SRP binds to the signal sequence and pauses translation, directing the ribosome to the SRP receptor on the ER membrane.
• Translocation initiation: The ribosome-SRP complex binds to the Sec61 translocon, and SRP is released. The ribosome resumes translation, pushing the growing polypeptide through the translocon.
• Soluble proteins: For soluble proteins, the entire polypeptide enters the ER lumen, and the signal sequence is cleaved by signal peptidase.
• Membrane proteins: For membrane proteins, the translocon integrates them into the ER membrane via hydrophobic stop-transfer sequences or start-transfer sequences that remain embedded in the membrane.
Outline the experimental set up of a sucrose density gradient experiment that is used to separate organelles in a centrifuge tube (after formation of microsomes via dounce homogenization), and draw a diagram of where organelles with high to low density would migrate to within the tube upon high speed centrifugation
• Sucrose density gradients are used to separate organelles based on their buoyant density. After homogenization of cells and formation of microsomes, the homogenate is placed in a centrifuge tube containing a gradient of sucrose (higher density at the bottom, lower at the top).
Steps:
- Prepare a sucrose gradient in a centrifuge tube (e.g., 10% to 60% sucrose).
- Place the homogenate containing fragmented organelles (microsomes) at the top of the gradient.
- Perform ultracentrifugation at high speed (e.g., 100,000g) for several hours.
- Organelles migrate through the gradient based on their density: high-density organelles (e.g., rough ER) migrate to lower layers, while low-density organelles (e.g., smooth ER) remain near the top.
Diagram (High to Low Density)
Golgi fragments (low density)
Smooth ER microsomes
Rough Er microsomes (high density)
Define what a microsome is and how the density of microsomes differs (or doesn’t differ) between organelles
A microsome is a small vesicle derived from endoplasmic reticulum that is produced by the fragmentation when cells are homogenized. ER breaks into fragments which reseal to form closed vesicles called microsomes. Microsomes differ in density depending on the origin of the ER (rough ER is denser due to ribosome attachment; smooth ER is less dense due to the absence of ribosomes).
Define what a translocon is
A translocon is a protein complex in the membrane of the ER or the inner membrane of the mitochondria that facilitates the translocation of newly synthesized protein into these organelles. Ex the Sec61 complex forms the core of the translocon and provides a channel through which polypeptides pass during co-translational or post-translational translocation.
Describe the difference between the rough and smooth ER
- Rough – the rough ER contains ribosomes, conducts protein synthesis.
- Smooth – smooth ER does not have ribosomes, conducts lipid synthesis
- Ribosomes- composed of protein and RNA. Free cytoplasm or rough ER. Site for protein synthesis.
List the targeting sequence/motifs that are associated with directing a protein to the ER, mitochondria, peroxisome, and nucleus
Targeting Sequences/Motifs for Directing Proteins:
Endoplasmic Reticulum (ER)
Signal Sequence: A short N-terminal sequence (usually 15-30 amino acids) that directs proteins to the ER. It typically contains a hydrophobic core flanked by positively charged residues.
Mechanism: The signal recognition particle (SRP) binds to the signal sequence and directs the ribosome-protein complex to the ER membrane, where the protein is translocated into the ER lumen or membrane.
Mitochondria
Mitochondrial Targeting Sequence (MTS): Proteins destined for mitochondria have an N-terminal amphipathic α-helix that is recognized by the mitochondrial import machinery.
The MTS directs the protein to the TOM complex (Translocase of the Outer Membrane) and then through the TIM complex (Translocase of the Inner Membrane).
Different signals guide proteins to specific mitochondrial compartments: outer membrane, inner membrane, intermembrane space, or matrix.
Peroxisomes
Peroxisomal Targeting Signal 1 (PTS1): A C-terminal tripeptide sequence, often SKL (Ser-Lys-Leu), that directs proteins to the peroxisome.
Peroxisomal Targeting Signal 2 (PTS2): A N-terminal nonapeptide sequence that targets proteins to the peroxisome.
These signals are recognized by Pex proteins that mediate the import of peroxisomal proteins into the organelle.
Nucleus
Nuclear Localization Signal (NLS): A short sequence rich in positively charged residues (lysine and arginine) that directs proteins to the nucleus.
NLSs can be monopartite (one cluster of basic residues) or bipartite (two clusters of basic residues separated by a few amino acids).
Proteins with an NLS are recognized by importins, which transport them through the nuclear pore complex into the nucleus.
List the targeting sequence/motifs that are associated with directing a protein to the ER, mitochondria, peroxisome, and nucleus
Monitoring Protein Localization Using Imaging:
To monitor protein localization in cells, fluorescent microscopy techniques are typically used. Here’s how you can generally do it:
Tagging Proteins with Fluorescent Markers:
Fusion proteins: A gene encoding the protein of interest is fused with a gene encoding a fluorescent marker, such as Green Fluorescent Protein (GFP). This allows visualization of the protein within live cells or fixed cells.
The fluorescently tagged protein is expressed in the cell, and its localization is tracked using fluorescence microscopy.
Organelles Using the Secretory Pathway:
• Endoplasmic Reticulum
• Golgi Apparatus
• Lysosomes
• Plasma Membrane
• Endosomes
• Vacuoles (in plants/fungi)
Organelles with Their Own Import Machinery:
• Nucleus
• Mitochondria
• Peroxisomes
• Cytoskeleton (free ribosomes for protein synthesis)
