Module 4: Protein targeting Flashcards
Characteristics of peroxisome
- Bound by single membrane
- Responsible for oxidative and synthetic reactions in the cell.
- Does not have its own genetic information (i.e. genome).
- Reproduction by fission
Where is the protein catalase located in the peroxisome?
In the peroxisome, there are dense regions called protein aggregates which is almost exclusively composed of a protein called catalase.
Role of peroxisome in animal cells
cholesterol synthesis
Role of peroxisome in nerve cells
synthesize plasmalogen for cell membranes
Role of peroxisome in liver cells
site of oxidation of toxins (ex. alcohol)
Role of peroxisome in plant cells
convert fatty acids to carbohydrates which will then power seedling germination
Major function of peroxisome in all cells
breakdown of very long chain fatty acids through beta oxidation
What role does fatty acids serve in the body?
energy source
What is the unfortunate by-product of fatty acid metabolism by peroxisome?
hydrogen peroxide
Function of catalase
converts the toxic hydrogen peroxide into non-toxic oxygen and water molecules.
The abundance of catalase serves as a visual marker for peroxisomes in the cell
Advantage of using fluorescence over TEM
Fluorescence allows us to look a living cells
How are peroxisomes formed?
Through the division and incorporation of new proteins (division occurs through a process called fission)
Describe how peroxisomes are formed in detail.
- Peroxisomal proteins are synthesized in cytosol and then transported to peroxisome.
- Peroxisomal membrane proteins join precursor membrane to create a peroxisomal ghost.
- Once membrane proteins are present, they are used to transport peroxisomal matrix proteins (ex. catalase) into interior of its organelle.
- As more proteins are synthesized, the peroxisome can grow and new peroxisomes can form by a process called fission.
Luciferase
- an enzyme that serves as an excellent model for deciphering the mechanism of peroxisomal protein transport
- enzyme that allows bioluminescence that we see in fireflies
- found in peroxisomes of cells in the abdomen
What was the conclusion derived from the study in which luciferase was expressed in mammalian cells?
The luciferase went into the peroxisome of mammalian cells telling us that pathways for protein transport to peroxisome are conserved in fireflies to mammals.
Five rules for protein transport
- A signal sequence is on transported protein
- Receptor for that signal sequence is on target organelle
- Translocation channel is required to get the protein across membrane into organelle
- There is a requirement for energy at some step in the process.
- Needs to be a way of targeting a protein to specific and different locations within organelle
PTS1
- stands for perioxsomal-transport sequence 1
- signal sequence that was identified in luciferase and in many peroxisomal proteins in different species
- consists of tripeptide: serine, lysine and leucine or can be called SKL
- found at C-terminus of translated protein
Is PTS1 necessary for transport of proteins into peroxisome?
Yes
Is PTS1 sufficient to direct a protein in the peroxisome?
Yes
Pex5
- cytosolic protein that acts as a receptor and to the PTS1 sequence at the C-terminus of the target protein
- contains 7 TPR motifs that together form a PTS1 binding pocket
Wha makes up a TPR motfi?
two alpha helices and a loop
Can Pex5 bring the protein to the peroxisomal membrane on its own?
No it must associate with transmembrane Pex14 protein
How does the peroxisomal protein on the surface of the membrane get into the organelle?
It uses a translocation channel. There are two proteins that can form this channel.
Does Pex 14 have any other function than recognition for Pex5?
Yes. It forms a translocon channel that transports Pex5 with target protein into peroxisomal matrix. Pex5 then releases peroxisomal matrix protein and Pex5 is recycled back out of cytosol to be reused.
Peroxisomal matrix
interior of peroxisome
In what part of the process is energy used in peroxisomes?
- Pex5 is uniquitinylated (requires energy) to release the matrix protein and deubiquitinylated to recycle Pex5 out to cytosol
Two places that proteins can go to in peroxisome
matrix and membrane
Why is it that when there is a mutation in PMP70, catalase also remains in cytosol?
Because if system to transport peroximal membrane is faulty, as a side effect, the cell cannot transport proteins into matrix.
Zellweger’s syndrome
- autosomal recessive disorder caused by mutations in genes that encode peroxisomal transport proteins (Pex2, Pex3, Pex5, Pex 10, and Pex12)
- mutations in these lead to same phenotypes
- causes impaired peroxisome function which accumulates very long chain fatty acids (since it is normally degraded by peroxisomes)
- no treatment is available.
Characteristics of Mitochondria
- Bound by double membrane
- Primary site of ATP production
- Have their own DNA
- Reproduction by fission
Outer membrane
simply surrounds the entire organelle
Inner membrane
much more complex with involutions that create cristae
Cristae
- distinct regions within the matrix of mitochondria
- increases the surface area of inner membrane where ATP synthesis occurs
Intermembrane space
- region that lies between the inner membrane and outer membrane
Where is ATP created in the mitochondria?
Inner membrane using ATPase and proteins of electron transport chain
______ of mitochondria enables high levels of ATP synthase
Structure
How does mitochondria usually appear in images?
as an elaborated network of tubules
Fission
- one mitochondrion becomes two new mitochondria
- process in which new mitochondria is created
Fusion
- process in which two mitochondria become one mitochondrion
Four destinations in which proteins can be located
outer membrane, intermembrane space, inner membrane, and matrix
Two places where proteins come from
- Mitochondrial genome and are synthesized by mitochondrial ribosomes.’
- Coded by nuclear genes - genes are transcribed in nucleus, mRNA is translated by cytoplasmic ribosomes, and then transported to mitochondria in post-translational pathway. (major method)
Protease
enzyme that degrades proteins in solution
What is the signal peptide sequence for mitochondrial protein transport into the mitochondrion from the cytosol?
matrix-targeting motif
What receptor does the matrix-targeting motif bind to?
matrix-targeting motif is recognized by an import receptor on the outer membrane of the mitochondria
Tom40
- general import pore embedded in the outer membrane
- known as translocon of outer membrane
Tim44, Tim23, Tim17 complex
- translocon of inner membrane
Contact sites
allow direct movement through the translocons
What is the purpose of cytosolic Hsc70?
- keeps polypeptides unfolded because the mitochondrial proteins must be unfolded to fit through the pored of the Tom and Tim translocons
What process of post-translational protein transfer into mitochondria requires ATP?
the Hsc70 function requires ATP
What is the purpose of matrix Hsc70?
- to bring proteins to the matrix
- it binds to the polypeptide and helps pull it out of the translocon and prevents it from moving backwards
Two compartments in which proteins travel to in the mitochondria?
mitochondrial matrix and inner membrane
what are the two targeting motifs that are needed when a protein goes into the inner membrane?
- N-terminal matrix-targeting motif
2. Hydrophobic stop-transfer sequence
What is the function of the stop-transfer sequence?
- Stops translocation so that protein is no longer being pulled through translocon
- It directs transfer of polypeptide out of translocon and into membrane
Three things that defective import of mitochondrial proteins can arise from?
- mutations in targeting signal
- mutations that disrupt import machinery
- deficiencies in the chaperones
Protein transport to the mitochondria and peroxisomes occur ________.
post-translationally
Characteristics of RER
- Bound by single membrane
- Does not have its own genome
- Site of protein synthesis and modification
- First destination for secreted proteins
Why is a portion of the RER labelled “rough ER” instead of just ER?
it is labelled rough because a portion of the ER has a punctuate appearance created by associated ribosomes
Are proteins transported to the lumen of the ER immediately upon translation so that they are not exposed to the cytosol?
Newly synthesized proteins can move directly into the microsome without being exposed to external environment
Microsomes
created by disrupting the ER membranes of the cell in a process called homogenization
Does translocation and translation need to occur together?
Yes, they must occur co-translationally
