Lecture 9: Membranes as borderlines - 2 Flashcards
What is used as a signal for cells to induce certain processes?
The targeting sequence of proteins.
What does this targeting sequence mean?
This sequence acts as an “adress”. It determines the destination of the newly translated protein. Targeting sequences are recognized by receptors present on the acceptor compartment; this is true for membrane proteins but also for soluble proteins.
Explain how membrane structures have evolved.
- Many organelles have been formed by invagination of the plasma membrane. Here, the nucleus, E.R. and peroxisomes have been engulfed and surrounded by the plasma membrane.
- The mitochondria and chloroplast membranes are derived from bacteria (endosymbiotic theory). Therefore mitochondria and chloroplasts still have DNA and ribosomes to synthesize primarily membrane proteins.
Describe the function of the endoplasmatic reticulum (E.R.).
The E.R. is the start of the secretory pathway and is a major site of synthesis of the membrane (by synthesis of lipids and proteins). There are many ribosomes in the E.R.
Shortly describe the secretory pathway.
The E.R. with its ribosomes is located around the nucleus. Proteins and lipids that are synthesized in the E.R. are directed to the Golgi apparatus with the help of vesicles. From the Golgi apparatus, vesicles with cargo are directed to the plasma membrane (or lysosomes).
Describe the process of vesicle budding and fusion with membrane components as cargo.
In the donor compartment vesicles are formed. During this process of budding, content and membrane proteins are taken up in the vesicle. The vesicle can then fuse with the target compartment, where the target compartment now contains components of the donor compartment.
- This is a highly regulated process, where the right cargo must be selected (soluble and membrane) for budding and fusion. Folding and modification of proteins is done along the way.
What cell compartment is a central event in membrane biogenesis?
The endoplasmic reticulum
What is protein targeting/sorting?
The process by which proteins are transported to their appropriate destinations within or outside the cell.
Is the process of protein targeting the same in eukaryotes as prokaryotes?
Yes, the principle is the same where there are ribosomes that make proteins. Some of these proteins will stay in the cytosol (because they have a function in the cytosol) and some proteins get transported to the membrane.
- An interesting similarity is that the sorting of proteins from ribosomes to the inner membrane of bacteria is the same process of sorting proteins from ribosomes to the E.R. in eukaryotes.
What do proteins that are directed from the cytosol to the E.R. in eukaryotes and proteins that are directed from the cytosol to the inner membrane of prokaryotes have in common?
A signal peptide sequence → peptide with an N- and C-terminus and a hydrophobic core.
(Tell’s us that this mechanism is highly conserved)
What are the basic components of protein targeting, translocation and insertion?
Ribosomes, chaperones, receptor, translocon/channel, ATP
What are the basic mechanisms of protein targeting, translocation, and insertion?
- Ribosomes to synthesize proteins
- Synthesized proteins are directed to their final location (e.g. on the membrane or across the membrane).
- If the protein needs to be translocated across the membrane, you need a channel (translocon) due to the proteins’ hydrophilicity. The channel is small and only opens when it’s activated.
- To open the channel, the signal sequence needs to be recognized by the channel’s receptor and also energy (in the form of ATP) is needed to open the channel.
- Chaperones are needed for folding and unfolding of proteins.
Do folded or unfolded proteins pass the translocon more easily/better? What is used to unfold and refold proteins before and after translocation?
Unfolded proteins, because these have a smaller diameter and the translocon is very small. Therefore chaperones are used to un- and refold proteins.
What is co- and posttranslational processing of proteins that will translocate through the membrane?
- Posttranslational processing → protein is synthesized and binds to chaperones before it will translocate to the membrane.
- Co-translational processing → ribosome moves and binds to the membrane while the protein is being synthesized and is then translocated through the membrane.
Note: co-translational processing occurs the most.
What determines where the ribosomes will synthesize their proteins?
The signal sequence of the mRNA. If it contains the signal sequence for the E.R. (with the hydrophobic core), the ribosomes will translate the mRNA as a co-translational process.
- Describe the free ribosome cycle.
- Describe the membrane-bound ribosome cycle.
- There are free ribosomes in the cytosol (free polyribosome) that synthesize proteins that are needed for the cytosol (and thus contain a signal sequence for the cytosol).
Therefore in the cytosol, there’s a common pool of ribosomal subunits.
- This pool of ribosomal subunits can be used to synthesize proteins that are translocated to the E.R. Here the E.R. signal sequence is recognized by the ribosomes and Signal Recognition Particle (SRP) machinery. The mRNA is then translated on the membrane (cotranslational processing).
How does the SRP machinery stimulate cotranslational processing of proteins?
When the ribosome binds mRNA containing an E.R. signal sequence, this complex will bind to the SRP machinery. This stops further synthesis of the protein. The complex will search for the SRP receptor, which is close to a translocon. The SRP machinery gets released and protein synthesis is continued → co-translational processing.
Why do we study membrane proteins?
- 30% of all sequenced genes encode membrane proteins.
- Membrane proteins are involved in vital cellular processes (signal transduction, communication, transport).
- 70% of drug targets are membrane proteins.
- Many diseases exist due to compromised folding/processing of membrane proteins (Cystic Fibrosis, Alzheimer).
- Very few high-resolution structures are available.
Why do we use E. coli as model organism?
- Targeting and insertion of membrane proteins are conserved, so also present in humans.
- E. coli’s genetics are accessible for manipulation.
- In vitro assays for transcription/translation/insertion are possible.
- Widely used as host for membrane protein overexpression.
What differences and similarities are there between post- and co-translational processing of proteins?
- Difference → post-translational processing uses chaperones, while co-translational processing uses SRP machinery.
- Similarity → both use a translocon.
What differences is there in post- and co-translational processing of proteins between eukaryotes and prokaryotes?
Prokaryotes have two membranes (inner- and outer membrane), so sometimes proteins need to pass two membranes.
So the signal peptide sequence determines whether a protein is synthesized via post- or co-translational processing.
What happens when the signal peptide is strongly hydrophobic? And what happens when it is less hydrophobic?
- Stronlgy hydrophobic → co-translation processing. SRP machinery has a high affinity for signal sequences with a strong hydrophobic core. This makes sense, since membrane proteins need strongly hydrophobic proteins.
- Less hydrophobic → post-translational processing.
Describe the characteristics of the SRP machinery.
SRP consists of a large RNA molecule (scaffold) with 6 proteins arranged on it. The proteins all have different functions important for the total SRP machinery function.
- One protein binds to the signal sequence peptide.
- Some proteins are important in the arrest of translation.
- One protein is able to bind to an elongation factor binding site on the ribosome.
The SRP machinery can change conformation depending on the contact it has with other molecules. What molecules?
- Binding of the signal peptide induces a conformational change.
- This is governed by GTP binding and hydrolysis.
