Protein Targetting Flashcards
What is the primary function of mitochondrial targeting signal sequences?
To direct proteins to mitochondria for import
Mitochondrial signal sequences typically have a high content of Arg and Ser/Thr, are 20 to 80 amino acids long, and are usually cleaved after import.
What is the structure of mitochondria?
Organized into four separate compartments
- matrix
- inner membrane = impermeable
- outer membrane = permeable to small molecules (requires transporters for larger ones)
- intermembrane space
What percentage of the proteome is targeted to the endoplasmic reticulum (ER)?
Approximately 30%
This includes both secretory and membrane proteins.
What are the two main types of proteins that enter the ER?
Soluble proteins
- initially imported from the cytosol into the ER and are then transported by vesicular transport to their final destination
- water soluble (aqueous proteins)
- secreted from the cell into extracellular environment (secretory proteins e.g. hormones & growth factors)
- located in the lumen of organelles of the endomembrane system (e.g. ER, Golgi, lysosomes)
- Examples include insulin, digestive enzymes, lysosomal enzymes
Integral membrane proteins
- hydrophobic sequences that form membrane spanning domains
- embedded into the lipid bilayer and are then transported by the vesicular system
- membranes of endomembrane system, plasma membrane but NOT mitochondria
- Examples include membrane receptors, channels, transporters
What is the role of chaperone proteins (Hsp70s) in mitochondrial protein import?
To pull proteins into the matrix and help refold them
This process is driven by ATP hydrolysis.
What is the difference between rough ER and smooth ER?
- Rough ER: Has bound ribosomes for protein synthesis
- Smooth ER: Involved in lipid synthesis
What is the common feature of ER signal sequences?
Stretch of 8+ hydrophobic amino acid residues usually near the N-terminus
Amino acids = valine, isoleucine, leucine etc
An example is the ER signal sequence from human insulin.
What initiates the pause in translation during ER targeting?
Binding of the signal recognition particle (SRP) to the ER signal sequence
This allows the ribosome to be directed to the ER membrane.
What type of translocation occurs when proteins enter the ER?
Co-translational translocation
This means that translocation occurs simultaneously with translation.
True or False: Most mitochondrial proteins are encoded by mitochondrial DNA.
False
Most mitochondrial proteins are encoded by nuclear genes and imported from the cytosol.
What is the function of the Sec61 channel?
To translocate proteins across the ER membrane through a central pore
The Sec61 channel is a multi-subunit complex that forms a channel in the ER membrane
Pore is closed in the absence of a translating polypeptide to maintain permeability
Fill in the blank: Mitochondrial targeting signals are usually located at the _______.
N-terminus
What is the energy source used during mitochondrial protein import?
ATP
This energy is required for the action of chaperones and translocators.
What is an amphiphilic α-helix in the context of mitochondrial signal sequences?
A structure where positively charged residues cluster on one side and nonpolar residues on the other
This structure is important for the function of the signal sequence.
What is the main purpose of the endoplasmic reticulum (ER) in eukaryotic cells?
To serve as an entry point for proteins destined for secretion and other organelles
What drives the import of mitochondrial proteins?
ATP hydrolysis by chaperone proteins
Location of mitochondria
Near sites of high ATP utilisation e.g. cardiac muscle
Structure of mitochondrial targeting signal sequences
High content of Arg (positively charged) and Ser/Thr (polar & hydrophlic but NOT charged)
At the N terminus
Between 20-80 amino acids long
Usually cleaved after import into the mitochondrial matrix
How are mitochondrial proteins unfolded during import?
- Mitochondrial targeting signal is recognised by a receptor in the outer membrane of the mitochondria
- Translocator channel (TOM) moves the protein to the inter-membrane space
- Signal binds a second translocator (TIM) in the inner membrane to move the protein into the matrix
- Signal is cleaved to allow the protein to fold into the right conformation
Distribution of ER in different cell types
Amount of rough & smooth ER varies depending on the requirement for protein and lipid synthesis
Pancreatic cells = synthesise insulin and have extensive rough ER
Hepatocytes (liver cells) = extensive smooth ER
Describe what is meant by co-translational transport of proteins in the ER
Ribosomes synthesising proteins without a signal sequence remain free in the cytosol
Ribosomes synthesising proteins with an ER signal sequence are directed to the ER while the protein is still being translated by the ribosome
Describe the role of SRP in targeting to the ER
- SRP (signal recognising particle) recognises hydrophobic residues in the signal sequence
- SRP in the cytosol binds the ER signal sequence as it emerges from the ribosome = translation is paused
- SRP binds to the SRP receptor in the ER membrane
- SRP is displaced and released for reuse
- The ribosome passes to the translocator (Sec61) = translation is resumed
How are soluble proteins released into the ER lumen?
- ER signal sequence binds to Sec61 translocator = channel opens
- Polypeptide is threaded through the channel as a loop while translation is still occurring = co-translational translocation
- Signal sequence is cleaved by signal peptidase
- Protein is released
