Week 6 Flashcards
When is the topology of the ER membrane established?
The topology of integral membrane membrane proteins in the secretory and endocytic pathways is established during insertion into the ER membrane and is maintained as the protein is transported to other membranes.
How is the topology of the ER membrane established?
Topology is established by topogenic signals in the polypeptide and the ER translocation machinery
What translocon is required to make the membrane proteins for the ER, nuclear envelope, Golgi, plasma membrane, endosomes, and lysosomes?
The SRP-Sec61 translocon
Topology
Topology refers to the orientation of the protein with respect to the membrane and the cytosolic / lumenal spaces. This includes the number of transmembrane segments, the orientation of the N- and C-termini and the where the side of the membrane where each loop between transmembrane segments is found.
PCC
Protein conducting channel
NC
Nascent chain
Composition of protein transmembrane domains
Transmembrane domains typically have ~20 hydrophobic amino acids flanked by charged residues
Stop-transfer sequence of single-pass transmembrane protein with a cytosolic C-terminal tail
The stop-transfer sequence is hydrophobic and becomes the transmembrane domain of this protein
Start-transfer sequence of a single-pass transmembrane protein with a cytosolic N-terminal tail
Often called an uncleaved signal sequence of a signal-anchor. The start-transfer hydrophobic sequence with flanking charged residues are topogenic signals
How does charge in the signal sequence determine the orientation of a transmembrane protein?
The positive (in) charge will be on the cytosolic side and the negative (out) charge will be in the ER lumen.
How are transmembrane segments detected using a hydropathy plot and computers?
For the hydropathy plot, a computer program will scan along the amino acid sequence of a protein calculating the average hydrophobicity of 21 residue “windows”. 21 is often chosen for the window size because it takes around 20 amino acids to span the bilayer (assuming an alpha-helix for the 20 residues).
What type of experiment can be performed to assess protein topology?
Protease protection assays. The sample is subjected to SDS-PAGE to determine which protein bands shifted in mobility or disappeared.
N-linked glycoprotein biosynthesis in the ER and Golgi is associated with which amino acid?
Asparagine (Asn) (N)
O-linked glycoprotein biosynthesis in the ER and Golgi is associated with which amino acid?
Serine (Ser) and threonine (Thr)
Where does the energy required for N-Glycosylation come from?
This is a biosynthetic process. Energy is required and comes from nucleotide triphosphate hydrolysis during formation of sugar-nucleotide donors.
Congenital disorders of glycosylation (CDGs)
Congenital Disorders of Glycosylation (CDGs) are a large set of rare genetic diseases caused by defects in the glycosylation of proteins. lead to defects in multiple organ systems and more severe cases are lethal.
Production of an N-linked oligosaccharide
The oligosaccharide precursor is transferred en bloc from dolichol to Asn residues in the sequence Asn-X-Ser/Thr by oligosaccharyltransferase (OST). This produces an N-linked oligosaccharide.
After being transferred to a growing polypeptide chain, what happens to the N-linked oligosaccharide?
It is immediately trimmed. These glycosylation and trimming events occur only in the lumen of the ER and are common to “all” eukaryotes.
Mutations in glucosidase II results in what disease in humans?
Mutations in glucosidase II causes polycystic liver disease in humans.
Quality control mechanism in the ER for recognizing unfolded glycoproteins
A glycosyltransferase recognizes unfolded glycoproteins and reglucosylates the N-linked oligosaccharide.
Calnexin
A folding chaperone that recognizes unfolded proteins bearing monoglucosylated N-linked oligosaccharides.
Protein chaperones that work with calnexin and calreticulin to fold proteins in the ER lumen.
BiP, an Hsc70 ATPase, and Protein Disulfide Isomerase (PDI)
What helps ensure the correct disulfide bonds form in the ER lumen?
The ER lumen is an oxidizing environment and proteins inserted into the ER often have multiple disulfide bonds between Cys residues. PDI and similar enzymes help ensure that the correct disulfide bonds form.
Why are disulfide bonds rare in cytosolic proteins?
Disulfide bonds in cytosolic proteins are rare because the cytosol is a reducing environment.
What happens to permanently misfolded proteins?
Permanently misfolded proteins are subjected to ERAD (ER associated degradation).
Process of ERAD
The glycoprotein is retro-translocated (dislocated) through a channel and degraded by the proteasome. This is the fate of many membrane proteins misfolded because of genetic mutation (Cystic fibrosis).
Which type of proteins are preferentially selected for ERAD?
Misfolded glycoproteins missing one mannose (by the action of ER mannosidase) are preferentially selected for ERAD. The ER mannosidase acts slowly and so only proteins staying in the ER for a long time will have the this mannose removed.
UPR
The unfolded protein response
Proteins involved in budding
Coat proteins and small GTPases
Proteins involved in transport
Cytoskeleton and motor proteins
Proteins involved in targeting
Tethers and Rabs
Proteins involved in fusion
SNAREs and NSF
The biosynthetic pathway
The biosynthetic pathway for sorting of newly synthesized lysosomal enzymes follows the secretory pathway through the ER and Golgi before passing through the late endosome on the way to the lysosome.
Two functions of coat proteins
- Deformation of the membrane into a vesicle
- Selection of cargo that is incorporated into the vesicle.
The vesicular transport steps are mediated by what three things?
COPI, COPII, and clathrin
Four steps of budding of COPII vesicles from the ER
- Recruitment and activation of Sar1 by the Sar1 receptor and GEF
- Recruitment of the cytosolic COPII components to the ER by activated Sar1
- Assembly of the coat imparts curvature to the membrane
- Cargo proteins are selected by their ability to bind COPII subunits
How is curvature induced in the ER membrane?
Insertion of the Sar1 amphipathic helix into the membrane expands the outer leaflet of the membrane and induces curvature in the membrane. The curved membrane is stabilized by Sec23/24 interaction with the membrane.
Inner coat of COPII
The inner COPII coat (Sec23) is the Sar1-GAP
Why is it essential that the COPII coat dissociates from the membrame?
After the coated vesicle forms, it is essential that the coat dissociates from the membrane so the COPII subunits can be recycled and the vesicle can fuse with the Golgi.
Exit signals for ER to Golgi transport
Exit signals are simple 2 or 3 amino acid sequences
VTCs
Vesicular tubular clusters
Which direction do VTCs go in?
On their microtubule-dependent transport to the Golgi, VTCs use minus-end directed motor protein (dynein).
The KDEL receptor and COPI vesicles are involved in what process?
Golgi to ER retrograde protein transport
How are escaped ER proteins retrieved?
- Occasionally, ER proteins are mistakenly incorporated into COPII vesicles for the trip to the Golgi complex.
- Residents of the ER lumen (soluble proteins) have a C-terminal KDEL sequence that is a retrieval signal
- The escaped proteins bind the KDEL receptor in the Golgi and are packaged into COPI vesicles for the return trip to the ER
ARF
Small GTP binding protein
COPI cargo
Cargo are ER proteins, the KDEL receptor, SNAREs and perhaps Golgi proteins
What determines if the KDEL receptor will bind or release its ligand?
Going from the ER to the Golgi, the environment gets increasingly acidic. The shallow pH gradient is established by the V-ATPase and may be responsible for determining whether the KDEL receptor will bind or release its ligand.
What type of motor proteins does COPII use?
Dynein motor proteins
What do vesicles budding from the ER associate with?
Vesicles budding from the ER fuse to form VTCs and then associate with a minus-end directed microtubule motor (dynein).
What do vesicles budding from the Golgi associate with?
Exocytic vesicles budding from the Golgi associate with a plus-end directed microtubule motor (kinesin).
Rabs
Small GTP-binding proteins that ensure vesicles interact with the correct tracks (motor protein), tethers and SNAREs
The TRAPP complex
Both a vesicle tether and a GEF for Rab2
Role of PI 4,5 bisphosphate with Rab
Both of these help determine the identity of membranes so transports are brought to the correct location.
When the Rab is in its GTP-bound form, what will it bind to?
The tethering factor, which leads to the capture of the vesicle from long distance, bringing it toward the membrane.
Location of v- and t-SNAREs
Vesicle will have V-snare proteins and target membrane will have T-snare proteins
What type of processes are used to study SNAREs?
Biochemical processes
The SNARE hypothesis
- Complementary sets of v- and t-SNARES operate in specific transport pathways.
- There are more than 20 different SNAREs operating in the secretory and endocytic pathways
What do SNAREs form from?
SNAREs form a four helix bundle that drives membrane fusion. For some transport steps, three SNAREs can generate the four helix bundle, other steps require four SNAREs
Botulinum toxin (BoTox) and Tetanus toxin
Botulinum toxin (BoTox) and Tetanus toxin are proteases that specifically target SNAREs that drive fusion of synaptic vesicles to the presynaptic membrane.
Role of SNAP25
SNAP25 is a t-SNARE involved in membrane fusion. SNAP25 is palmitoylated - this fatty acid facilitates membrane association.
Source of energy that drives membrane fusion
When the amino acid termini of the four-helix bundle comes together, they “zip up.” When proteins bind, energy is released, and this is the source of energy in this process.
Why do SNAREs zip up?
Zippering up of the SNAREs is thought to pull the apposing membranes together to exclude water and drive fusion of the bilayers.
Hemifusion intermediate
Fusion proceeds through a hemifusion intermediate where the cytosolic leaflet fuses before the lumenal leaflet.
Steps in SNARE-mediated fusion of two bilayers
- Tethering proteins help target and dock the vesicles on the appropriate membrane.
- SNAREs then associate and pull the two membranes into close proximity (excluding water in the process)
- The membrane bilayers are destabilized at the point of contact allowing exchange of phospholipid molecules between the vesicle membrane and target membrane.
What protein drives dissociation of the cis-SNARE complex?
An ATPase called NSF
What happens to the v-SNARE involved in fusing COPII vesicles with the cis Golgi?
The v-SNARE involved in fusing COPII vesicles (or VTCs) with the cis Golgi are recycled back to the ER in COPI vesicles.
What happens to the v-SNARE involved in fusing COPI vesicles with the ER?
The v-SNARE involved in fusing COPI vesicles with the ER is recycled back to the Golgi in COPII vesicles.
How does NSF work to reset the cis-SNARE complex?
NSF jumps onto the cis-SNARE complex and pries it apart using ATP hydrolysis. Puts energy in to separate the proteins so new trans-SNARE complexes can be formed
Role of ubiquitination in vesicular transport
- Ubiquitination drives COPI priming and Golgi SNARE localization.
- Ubiquitination turns off SNARE, returns it to its target, and then ubiquitin is removed so the SNARE can be activated.
Cis face of the Golgi
The forming face of the Golgi. This is where COPII binds.
Trans face of the Golgi
Vesicles bud off the trans face and go to the plasma membrane.
Importance of sialic acid on glycoproteins (modified in the Golgi)
Sialic acid terminates the glycoprotein chain. The sialic acid humans produce is chemically different from the sialic acid produced by other mammals. This is an important way the immune system distinguishes self from non-self.
Endoglycosidase H (Endo H)
Endoglycosidase H (Endo H) is a commercially available enzyme that is used experimentally to determine if a glycoprotein carries high-mannose or complex oligosaccharides. The presence of complex, Endo H-resistant N-glycans indicates that the protein has reached the Golgi complex.
CFTR-∆F508 experiment
This experiment provides the evidence that the band marked “B” is CFTR bearning at least one N-linked oligosaccharide. The fact that Endo H treatment causes a shift in the mobility of the “B” form of CFTR is conclusive evidence that it has a “core” (meaning ER form) N-linked oligosaccharide. The form marked “C” has received Golgi modifications and is mostly resistant to endo H.
Where does the proteolytic processing of many different prohormones occur?
Either in the trans-Golgi network (TGN) or in secretory granules.
What catalyzes the proteolytic processing of prohormones?
Prohormone convertases (proteases)
