MP3: How are molecules moved in cells? Flashcards
Why is it easier to crystallize a thermophile protein?
When you reduce the temperature, it automatically becomes less ‘chaotic’.
What is the aqueous channel hypothesis?
Proposes that ion channels form narrow aqueous pores through the membrane, allowing the selective transport of ions that is much faster than simple diffusion.
Describe the structure of the Sec translocon.
- Conserved across species (although subunits have different names)
- Sec61a/SecY is the main structural component
- Channel constricts to form a pore ring
- Plug helix sits at the bottom underneath the pore ring.
- Lateral gate between helices 7 and 2.
How can a homogeneous population of ribosome-nascent chain complexes be produced?
Remove the STOP codon. The halts the ribosome and thus every mRNA missing the STOP codon will have a ribosome at exactly the same chain position.
What is the loop model of protein insertion?
A mechanism for how membrane proteins are inserted and integrated into the lipid bilayer of the membrane via the Sec translocon.
Signal peptides from the NCC bind to the translocon in an inverted orientation i.e., N binds at a cytoplasmic side of the translocon, but C keeps being pushed through to the lumenal side of the membrane.
As the ribosome keeps producing polypeptide, a loop is extruded that continues until signal peptide peptidase (SPP) cleaves the signal peptide off.
How is the Sec translocon lateral gate opened for protein insertion?
The channel becomes primed for protein translocation upon ribosome binding, triggering conformational changes that crack the lateral gate (i.e., no translocating polypeptide is yet present).
The signal peptide then positions itself where helix 2 normally sits, practically wedging the gate fully open.
With the gate open, interactions between the channel walls and the plug are weakened, and the plug is displaced from the channel.
What is the general structure of a signal peptide?
N-terminus: positive
Core: hydrophobic
C-terminus: cleavage site
What is the positive-inside rule?
Positively charged amino acid residues (such as Lys or Arg) are preferentially located on the cytoplasmic side of the membrane, whilst negatively charged residues (such as Asp or Glu) are located on the extracellular side.
What is a signal anchor sequence?
A membrane targeting signal found in some TM proteins. They differ from other signals as they don’t require cleaving to release the protein. Instead, the sequence is incorporated into the final protein structure.
How does co-translational translocation differ to post-translational translocation?
Co-translational:
- Signal peptide is recognized by the SRP
- SRP halts further translation (in eukaryotes) and directs the complex to the SRP receptor on the plasma membrane (prokaryotes) or ER (eukaryotes).
- Physical docking of the ribosome
- Signal peptide insertion
Post-translational:
- Mostly only in prokaryotes
- Signal peptide is recognized by SecB
- SecB transfers the peptide to SecA ATPase
- SecA pumps the protein through the translocon
- Usually much shorter proteins
Why don’t ions leak through the Sec translocon?
Normally, the pore ring and plug block leakage. When the plug domain is removed by a translocating polypeptide, the pore ring seals around the polypeptide, so there isn’t enough space for ions to get through.
The hydrophobic environment of the pore ring discourages desolvation of ions by imposing a net energetic cost to removing the water molecules, giving them an even large hydration radii.
Why are proteins usually transported unfolded? What is the potential con of this method?
- Easier to maintain a seal around an unfolded protein
- Cross-sectional area is much smaller when unfolded
- Transport is independent of the final fold of the protein
Mechanisms are required to keep the substrate protein unfolded.
What energy sources are available to actively transport proteins across:
1. Bacterial cytoplasmic membrane
2. Bacterial outer membrane
3. Mitochondrial inner membrane
4. Mitochondrial outer membrane
- NTPs and PMF
- No direct energization
- NTPs and PMF
- NTPs
Explain an experiment that can be used to assess whether a protein is being transported through a translocon.
Vesicles containing translocons are bathed in solution containing proteins that are being assessed for transport.
After a period of time, proteases are added to the solution. These will destroy any proteins that haven’t been transported into the vesicle. Analysis is via gel electrophoresis.
Where does the energy come from for co-translational protein transport?
The energy is the same energy used for protein synthesis and polypeptide elongation, which pushes the polypeptide through the channel.
This energy has already been spent to do translation, so translocation requires no extra energy.
What is the ‘-1/-3’ rule?
The signal peptidase recognition motif consists of small side chain amino acids (most commonly alanine) at positions -1 and -3, relative to the cleavage site.
Describe the process of the SRP targeting and binding the ribosome.
- SRP recognizes the signal sequence and binds the ribosome in its GTP-bound state.
- Binding causes translational arrest.
- SRP targets the ribosome to the ER and associates with the SR receptor.
- Nascent chain is directed into the translocon via GTP hydrolysis.
- Translation restarts.
What type of protein is the SRP? What are the two key domains on it?
Ribonucleoprotein
- Nucleotide-binding domain (for GTP)
- Recognition domain (for signal sequence)
How do post-translational and co-translational signal peptides differ?
Post-translational h-region is less hydrophobic.
What is post-translational translocation typically only of short proteins?
SRP recognition requires around 60 amino acids to be synthesized. If the chain is shorter than this, translation will have terminated prior to SRP recognition and so a mechanism is required to overcome this.
What are the two proposed models for BiP translocating a post-translational protein through the Sec translocon?
Brownian Ratchet: BiP binding prevents the polypeptide going back through the channel, and so Brownian motion predicts the chain will eventually translocate through the channel.
Power stroke: BiP binds the unfolded polypeptide and uses ATP hydrolysis to physically pull the chain through.
To complete the cycle, BiP must be removed which requires ATP hydrolysis.
How does protonmotive force assist Sec transport in bacteria?
SecDF is partially embedded in the membrane, and this membrane-embedded part can conduct protons.
This may be coupled to a conformational change in the periplasmic domain, by which a translocating polypeptide is bound and released.
NB: this only accounts for some of the dependence of protein translocation on PMF - we don’t fully understand the rest.
How did electrophysiological experiments show that precursor proteins are transported through a gated aqueous channel?
Two compartments were separated by a planar lipid bilayer that contained a single translocon. A potential difference was applied across the membrane and measurements taken to deduce the mechanism.
- Ions are unable to freely pass through the translocon when a nascent chain is being translocated.
- Puromycin incorporation into the carboxy-terminus of the nascent chain. Peptidyl-puromycin is released from the ribosome and translocated across the bilayer. This unplugs the protein-conducting channel allowing ions to pass freely through.
- Raising the salt concentration releases the ribosome from the membrane allowing the channel to close.
Showed that the ribosome has a function in keeping the channels open.
[Puromycin is a tRNA analogue that causes premature chain release.]
How are membrane proteins targeted co-translationally if they don’t have signal peptides?
They contain long stretches of hydrophobic amino acids (TMHs) that are used for targeting.
What is the lipid partitioning model?
This explains how a TM protein enters the bilayer, whilst soluble proteins don’t.
A TM protein within the aqueous channel will prefer to be in the hydrophobic binding site of the lateral gate, and even more so to be within the bilayer.
A soluble protein will want to remain in the aqueous channel and hence be transported all the way through.
What is the role of YidC/Oxa1 proteins in membrane protein insertion?
These TM proteins can act in two ways:
- Sec-independent: they can insert membrane proteins without Sec, but only very small proteins.
- Sec-dependent: they act as a chaperone to assist in the folding of Sec-integrated membrane proteins, especially for multipass proteins.
What are multipass proteins and why are they harder to insert into the membrane? How is this overcome?
They’re membrane proteins that span the lipid bilayer multiple times, often containing hydrophilic cores.
Although in the assembled state the channel the hydrophobic on the outside, hydrophilic amino acids can make insertion harder.
YidC/Oxa1 protects these polar groups until the 3D structures can be assembled.
TMCO1 and the PAT complex may also be required.
What structural features suggest that YidC is an insertase and not a transporter?
YidC contains a functionally important polar cavity for protecting hydrophilic amino acids.
This is only open to the cytoplasm and membrane interior, not the periplasm, which prevents the protein from acting as a transporter.
How are tail-anchored proteins inserted into the membrane?
The SRP signal for TA proteins is at the C-terminus, hence they are post-translationally transported.
They’re then inserted by Get1 complexes containing YidC homologues that lack the large polar domain.
What happens if the N-terminus (which normally contains the positive signal peptide) needs to be on the outside of the the membrane?
In this conformation, the N-terminus cannot open the lateral gate, so it’s instead inserted by a eukaryotic YidC homologue (EMC).
