User:Kinkreet/Protein Science/Get3

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The guided entry of TA proteins (a.k.a. Golgi ER trafficking or GET) pathway is a chaperone system that transport tail-anchored (TA) membrane proteins to the endoplasmic reticulum (ER). One of the key players of the GET pathway is the Get3 ATPase. In its free form, it exists as a dimer, although to function, it forms a tetramer.

Background (Daniel)
Membrane proteins must have a mechanism of insertion into the membrane after or during translation. The signal recognition particle (SRP)/Sec61 translocon pathway delivers most of the nascent membrane proteins to the endoplasmic reticulum (or the plasma membrane in prokaryotes, which lacks the ER) while the protein is being translated. The SRP binds to a signal sequence of a newly synthesized polypeptide, and also the SRP receptor on the ER membrane. This brings together the ribosome-nascent polypeptide complex to a channel on the ER called the translocon, which is close to the SRP receptor. Proteins inserted this way have their N-terminus in the lumen of the ER, or the extracellular side of the plasma membrane.

The transport inner membrane/transport outer membrane (Tim/Tom) translocases perform the same function, but with the mitochondrial membranes instead. Other pathways may exists, as TA proteins are found also in teh nuclear envelope and peroxisomes. Some proteins, such as cytochrome B5, do not require any special machinery for insertion

Tail-anchored (TA) proteins have their C-terminal domain, or at least a domain near the C-terminus, inserted into the membrane, with the N-terminus being in the cytosol. Tail-anchored (TA) proteins cannot be inserted using the SRP pathway, because it lacks a signal sequence, and so the SRP cannot bind. Instead, it has a hydrophobic region near its C-terminus, which will be inserted into the ER membrane post-translationally, using different pathways, of which the most common one being the guided entry of TA proteins (GET) pathway. Although this pathway is not the major pathway, it is found in all three domains of life still accounts for 2-3% of the open reading frame (ORF) in humans or about 5% of all membrane proteins and 1% in yeasts and prokaryotes. Vesicle/membrane fusion proteins, SNAREs, the apoptosis-related protein family Bcl-2 are all TA proteins. TA proteins usually have cytoplasmic functions, and some help identify the compartment as a certain type of organelle. It has been suggested that TA proteins can recruit lipids to form organelles.

The prompt insertion into the membrane is important because without insertion, the hydrophobic regions will aggregate.

Overview of GET pathway
Sgt2/Get4/Get5 (in yeast) or Bag6 (mammals) sorting complex delivers a TA protein to the ATPase Get3 (yeast) or TRC40 (mammals), which binds to the transmembrane domain of the TA protein and target it for insertion using Get1/Get2 receptor. The three Get proteins physically associates. Get1/2 is essential for TA protein insertion, and its absence causes a cytoplasmic accumulation of Get3/TA protein complex. Furthermore, the genetic deletion of any of the Get genes led to the loss of retrograde trafficking from Golgi to the ER membrane. The hydrophobic region is uniform for all TA proteins (they serve the same role), but after insertion into the ER membrane, there are more specific segments flanking the TMD which determines the sorting of the TA proteins to specific compartments within the cell. These signals may not be specific, because certain TA proteins, such as Bcl2, are found both on the membranes of mitochondria and the ER. Without Get3, the TA protein risks being inserted into the mitochondria, or other membranes.

Get3
The Get3 protein is the protein which binds to newly synthesized TA proteins and transport them for insertion. As a dimer, it can exist as an 'open' (no bound nucleotide) or 'closed' (ATP or ADP•AIF4--bound); this is usually at an equilibrium. When ATP is bound, the equilibrium position is shifted to the closed state; Get3 is now available to associate with Get4 and displays a hydrophobic groove which binds the TA proteins. After binding of the TA protein, the dimers dimerize to form a homotetramer which is then capable of insertion into the ER membrane. Get4 dissociates and Get1/2 associates with Get3, and facilitates the insertion of the TA protein. Suloway suggests that Get2 binds Get3/TA complex while Get1 disrupts this interaction to facilitate the dissociation of the TA protein, making it capable of insertion. Get3 is freed and return to being a dimer, ready for binding TA proteins again.

Questions
Why do TA proteins not have a signal sequence? are they less evolved?

Cloning, expression, purification and crystallization (Charline)
The Get3 genes were inserted into E. coli, it was then purified and crystallized in the presence of ADP or ADP⋅AlFX, forming two space groups.

Cloning
For the recombinant expression of all Get3 homologues the same technique was used. After a genomic DNA amplification for MjGet3 (MJ_1142) from M. jannaschi DSM 2661, TkGet3 (TK_0994) from T. kodakarensis KW128, MmGet3 (MmarC7_1163) from M. maripaludius C7 and ScGet3 from S. cerevisiae, the genes were cloned into pET33-b(+) vector. The vector was a bit different from the Novagen original vector as it was modified to express only a N-terminal poly-histidine tag (x6 his residues) for a later purification. Some MjGet3 proteins were modified by site directed mutagenesis to obtain two different truncation-encoding amino-acids; from amino acid 12 to 349 for one (MjGet312-349), and from amino acid 12 to 333 for the other (MjGet312-333).

When co-expression was needed, the vector pACYCDuet was used as the cloning vector for the Get3 homologues (either MjGet312-349, TkGet3 and ScGet3). This Novagen vector contains two Multiple Cloning Sites and the gene for the protein of interest (Get3) was cloned in the first MCS. Many TA proteins were used; Ysy YBR162W-A, Sbh1 YER087C-B, Secb, SecE MJ_0371, MtrA MJ_0851, MtrB MJ_0850. The genomic DNAs were cloned in a pMAL C2 modified vector. This vector usually used for protein Fusion and Purification was modified to contain a cleavage site for a thrombin protease between the Mannose Binding Site and the Multiple Binding site.

Expression
BL21-Gold (DE3) competent cells derived from E.coli cells were transformed with the vectors containing the Get3 homologues gene. These high-performance competent cells are based on the use of the T7 RNA polymerase promoter and lack (naturally or by engineering) the Lon protease and the OmpT protease, which save the recombinant proteins from degradation. The full BL21-Gold(DE3) genotype is E. coli B F– ompT hsdS(rB– mB–) dcm+Tetr gal λ(DE3) endA Hte. For the Get3 proteins to be expressed, the transformed BL21-Gold (DE3) were incubate during 3 hours in 2xYT at 37°C in the presence of 0.3mM Isopropyl β-D-thiogalactoside at an induced optical density of 0.6 at 600nm.

After resuspension in Buffer A (composition: 50 mM Tris pH 7.5, 300 mM NaCl, 10 mM b-mercaptoethanol) containing protease inhibitors, the cells were lysed through an ML-110 Microfluidizer. This microfluidizer disrupts the cells by application of high pressures. The soluble supernatant was obtained by centrifugation of the lysate.

For co-expression between ScGet3, MjGet3 or TkGet3 and TA proteins (see details in the cloning part) the same protocol was followed using the same strain BL21-Gold (DE3).

Purification
The obtained supernant was loaded on a Ni-NTA resin column for isolation and purification of the Get3 homologues. Actually being tagged with a 6xHis-tag, the Get3 homologues are able to bind the Ni ions of the column. The column was then washed with buffer A (see composition in Recombinant Expression Part) and 10mM imidazole. Finally the protein of interest was eluted using the same buffer A with 200mM imidazole. Indeed, imidazole displaces Histidine residues from Ni ions. After an incubation of for 16 hours at room temperature with 2U thrombin/ml in the same time as dialysing against buffer A, the eluate was passed over the same resin as before to only select cleaved and purified product. Then, a Superdex 200 column was then used to purify the Flow Through. For co-expression between ScGet3, MjGet3 or TkGet3 and TA proteins, the soluble complexes were purified in two times using two different types of resin in the column, an amylose resin column and a Ni-NTA resin. The amylose resin, composed of amylose and agarose beads, binds maltose-binding proteins and thus allows isolating TA proteins. The resulting eluate was then incubating overnight at room temperature with 2U of thrombin per ml to actually cleave the TA protein from the MBP.

Crystallization
To crystallise the MjGet3 proteins, two methods were used. MjGet312-349 crystals were obtained using the Sitting Drop Vapour Diffusion Method; with 1ml of 10mg/ml MjGet3 with 1ml of reservoir solution (see table for composition) after two days at room temperature. The crystals were in the P21 form. MjGet312-333 crystals were obtained using the same method but with 1ml of 10mg/ml MjGet3 (10mM Tris pH 7.5, 100mM NaCl, 10mM b-ME, 2mM MgCl2, 2mM ADP) with 1ml of reservoir solution (0.2M Na2SO4 and 10% (w/v) PEG 3350) after one. It crystallised in the P2 form.

For the crystal protection, MjGet312-349 crystals were cryoprotected with artificial mother liquor (20% glycerol, 17,5% sucrose, 17,5% xylitol) and then flashed freezed in liquid N2 whereas, MjGet312-333 crystals were cryoprotected with 20% ethylene glycol.

Data collection, structure solution and refinement
X-ray diffraction was used to elucidate the structure, using molecular replacement (MR) to solve the phase problem. MR aims to find a model which best fits the experimental electron densities of known structures, and so must have a search model. The nucleotide hydrolase domain (NHD) from the fungal Aspergillus fumigatus AfGet3 was used for this purpose. The final refined structure has R-factors of 29.6-28.6%, which is reasonable. The structures of the crystals (one with ADP and one with ADP⋅AlFX) are very similar, with a root-mean deviation of 0.8Å.

The structure elucidated was a homotetramer, ~150Å in length and ~60Å in diameter. The tetrameric nature is reinforced by size exclusion chromatography performed during the purification process.

Cysteine-coordinated zinc ion stabilizes the homodimeric interaction. And two such homodimers come together to forma tetramer. The monomers in the tetramer seem to be in the 'closed' form, where helix 7/8/9 are 'closer' to the NHD.

Pull downs
Get3 homologues (either ScGet3, MjGet3 or TkGet2) and TA proteins were co-expressed in BL21-Gold(DE3) competent E. coli. Bl21 competent cells are designed for high level of protein expression, using the T7 RNA polymerase promoter expression systems; they also lack OmpT and Lon proteases, which, if present, may degrade recombinant proteins. The gene which encodes for endonuclease I (endA), is also inactivated, to ensure the plasmid DNA is not degraded. The full BL21-Gold(DE3) genotype is E. coli B F– ompT hsdS(rB– mB–) dcm+Tetr gal λ(DE3) endA Hte.

The proteins are then purified using amylose resin (NEB)

the yeast homologue of mammalian Sec61b and a demonstrated GET pathway substrate

??
Both Get3 and the TA protein are tagged with affinity tags, this allows us to purify ScGet3 bound to different TA proteins, by using a different tag for each TA protein. The Get3/TA protein complex is analysed using inductively coupled plasma mass spectrometry (ICP-MS) to identify bound ions, and found there are one zinc ion to every two Get3.

One of the TA proteins used was Sbh1, the archaeal homologue of Sec61β. When Sbh1 is co-expressed, it was bound to be bound to Get3, indicated by the size of the band on the Western blot; as expected, Get3 expressed with a Sbh1 deletion did not show any form of complex at all, demonstrating the Get protein is specific to that one protein.

Using bioinformatics, proteins homologous to the known TA proteins were identified, purified and separated using affinity tags, and was found also to form a complex with Get3. Some of these homologues were found in M. jannaschii, such as SecE (a component of the Sec channel) and MtrA and MtrB (subunits of tetrahydromethanopterin S-methyltransferase. Both types of Get3 (those with a zinc coordinating cysteines, and those without) are able to bind all those TA proteins and their homologues.

The N-terminus of Sbh1 was fused with MBP, with a linker and thrombin protease cleavage site in between. This sequence was then co-expressed with ScGet3, which expresses with a 6xHis tag. The ScGet3/Sbh1 complex is then ran on a gel, with and without thrombin. If the thrombin cleavage site is within or close to the transmembrane domain, then addition of thrombin would not affect the length of the protein, because the cleavage site is protected by Get3. The transmembrane domain is estimated using TMHMM to be from residue 55 to 74, and from experiment, it is found to be around 52-82. This suggests that the hydrophobic groove only binds to the transmembrane domain and maybe a few residues either side of it, but nothing more.

ScGet3, when expressed in E. coli predominantly forms a dimer, but a small portion can also form tetramers, which overtime degrades into dimers. The reverse is not observed. Suloway hypothesize that the tetramer is stabilized by binding hydrophobic peptides produced by the E. coli, however, no evidence of such peptide was identified. TkGet3 is similar to ScGet3 and elutes both as a tetramer as well as dimer. MjGet3 and MmGet3 (from the mesophile M. maripaludis) are predominantly tetrameric in structure.

The majority of the interactions stabilizing the tetramer is attributed to the hydrophobic interactions of helix 8 of one subunit with helices 4 and 5 of the opposing subunit. They tested this by applying the detergent N-octyl-β-D-glucopyranoside (β-OG) just above the critical micelle concentration, for overnight. The majority of the tetramer dissociated into dimers.

Furthermore, they induced mutations to helix 8. The full and partial deletion of the whole helix resulted in loss of TA binding; two mutations at the 3-helix bundle interface (helices 8, and 4 and 5) - F192D and M196D resulted in dimer formation rather than tetramer; the mutation M193D, which is not at the interface, favoured the tetrameric, even though the mutation is one from a hydrophobic residue to a polar residue.

Microsome insertion assay
To test whether the Get3/TA protein complex is enough for insertion, a microsome insertion was carried out where purified ScGet3/MBP-tagged Sbh1 complexes were added to purified yeast microsomes. Microsomes were isolated from WT and Dget3 strains and resuspended in the reaction buffer (see composition in table 3). Then 10ml of this solution were mixed with 1ml of 2mM Get3 + 0.5 ml 100mM gluthatione + 1.2 ml 100mM batho cuprooine disulfonic acid + 1ml 100mM ATP + 14 ml of 2x ATPase Buffer (composition in table 3). The mixture was incubated at 30°C for 1 hour. A centrifugation step of the samples, in the presence of 490ml 50% Optiprep solution in the reaction Buffer, 1160ml 40% Optiprep and 450ml of the reaction buffer was following the incubation. The centrifugation was done during 3hours at 4°C at 166.000xg. The fractions were then collected and precipitated with 50% TCA. After two washing with cold acetone and drying at 37°C, the pellets were resuspended in 1xSDS Page sample Buffer. Once the microsomes were ready the insertion assays were realised with the purified Get3 complexes.

The results showed that it is indeed enough, signified by glycosylation of a opsin glycosylation site near the C-terminus of the TA protein, which can only happen if the TA protein is inserted with the C-terminus inside the microsome. If the microsome was disrupted, endoglycosidase EndoH deglycosylated the opsin site, confirming glycosylation is due to enzymes within the microsomes.

Nucleotide binding is required for insertion. When EDTA was added to disrupt nucleotide binding, no insertion was observed. Insertion also appears to be kingdom-specific, as MjGet3/MBP-Sbh1 was unable to insert into yeast microsomes. There are also no Get1/2 homologues in archaea, and so the mechanism for insertion might be different in different domains. Similar experiments was performed with fungal microsomes and no insertion was observed.

SEC-MALLS
SEC is a Size Exclusion Chromatography, also called Gel permeation chromatography, and MALLS (Multi Angle Laser Light Scattering) is a molecular weight detector. The purified proteins were passed through a Shodex KW-804 column (10mM Tris pH 7.5, 100mM NaCl, 10mM b-mercaptoethanol) using a MALLS detector to select them according to their molecular weights. Suloway was able to work out the relative molecular weight of the Get3/TA protein complex. The dimeric ScGet3 and the tetrameric MjGet3 had a relative molecular weights of 80.8 and 153 kDa. Knowing the molecular weights of the monomers, Suloway then tried to work the number of TA proteins that binds to each oligomer. He could not conclude but remarked that it is possible that multiple TA protein may be bound to a tetramer.

SAXS
Small-angle X-ray scattering (SAXS) is a technique that provides information about the structure of molecules without needed to grow crystals. In order use this method, the purified MjGet12-349 and ScGet3 complexes with TA substrats were dialysed.

Biological small-angle X-ray scattering (bioSAXS) was used to measure the dimensions of the complex, and thus able to confirm the dumbbell structure of the complex. It confirmed a multi-domain protein structure, which is similar in both fungal and archaeal tetramers.

Summary

 * The Get pathway is found in all domains of life.


 * The mechanism of insertion of archaeal TA proteins is likely to be different from fungal and eukaryotic insertions. Indicated because:


 * There are no Get1/2 homologues in archaeal
 * The archaeal TkGet3 is capable of TA protein binding and insertion, but does not have a CXXC motif, which is essential for insertion by fungal Get3.
 * The structure of archaeal Get3 is a tetramer.


 * The structure of fungal Get3/TA protein complex is a tetramer.


 * The dimer can be stable and is capable of TA binding using its hydrophobic groove, but only tetrameric Get3 was capable of insertion.


 * Helix 8 is essential for TA binding and insertion.


 * Helix 6 affects nucleotide binding, but is isolated from the hydrophobic groove, and so act to relay information from the groove to the nucleotide hydrolysis domain (NHD).


 * The binding of TA stabilizes the tetrameric conformation, and some does not require energy to bind.


 * The hydrophobic groove is flexible and can bind multiple TA proteins.


 * Nucleotide hydrolysis is not required for TA binding, but is required at the membrane for insertion.

Structure
Get3 can be a homodimer or a homotetramer.

Get3 is thought to serve as a mechanism by which TA proteins are inserted into different membranes on different organelles. Because prokaryotes have no such organelles, Get3 is thought not to exist in prokaryotes; indeed, none has yet been found. However, of the archaean genomes sequenced, hlaf have show to contain a Get3 gene. Get3 is especially prominent in the extremophiles - methanogens, halophiles and thermophiles - and suggests these organisms require specific membrane targeting of the TA proteins.

The crystal structure of a Get3 homologue has been elucidated in the archaea Methanocaldococcus jannaschii. The structure of a fungal Get3 bound to a TA protein was also elucidated using small-angle X-ray scattering, and shown that it is homologous to the achaean Get3 homologue.

Get3 consists of a ATPase domain, and a hydrophobic α-helical subdomain rich in methionine and glycine. "The ATPase domains form an extensive dimer interface that encloses 2 nucleotides in a head-to-head orientation and a zinc ion." The α-helical subdomain is highly flexible, and is used to bind to the TA protein.

Helix 6 lies at the base of hydrophoobic groove, and by hydrogen-deuterium exchange NMR, shows that the helix is more exposed after TA binding, as it moves closer to the dimer interface, to close the chamber to better fit the TA protein. Helices 4, 5, 7, 8 and 9 forms a central hydrophobic chamber (30Å across × 40Å up), and are in the same conformation TA-bound or unbound.

The disordered loop between helix 8 and 9 is required for separating the hydrophobic chamber from the cytoplasm, its absence gives a positively-charged opening.

Mutagenesis of the TA binding groove of ScGet3 showed that mutations near the center of the interior of the groove cause defects in binding, those near the edge/base of the groove (near helix 6) causes defects in ATP hydrolysis (as they are required to communicate the state of the chamber to the NHD), or both.

By performing a BLAST search, it appears there are no proteins similar to Get3, both at the genetic and protein level, suggesting that it is

Homologues
ArsA A structural related ATPase in bacteria used for arsenate export. It is a monomer that has a tandem repeat, so appears to be a pseudo-dimer. It also has a metal coordinating residue used for TA binding, with the same function as the Get3 motif of Get3.

Upon binding arsenate, the ArsA dimerize, to a structure homologous to the tetrameric MjGet3.

Most homologues usually have 2 conserved cysteines as CxxC to coordinate zinc, and is essential for function.

Function
When free, Get3 exists as a homodimer. When in this form, it can be in an 'open' or 'closed' configuration. In the 'closed' configuration, a helical subdomain (HSD) forms a hydrophobic groove that binds to the transmembrane domain of the TA protein.

The tetramer forms a hydrophobic chamber that allows the TA protein to bind, after which it is able to be inserted into the membrane.