Summary-Paper 34

Summary- paper 34: Lysosome transporter purification and reconstitution identifies Ypq1 pH-gated lysine transport and regulation

Felichi Mae Arines, Aleksander Wielenga, Olive E. Burata, Francisco Narro Garcia, Randy B. Stockbridge, Ming Li

Questions/gaps addressed:

Eukaryotic membrane proteins are usually expressed at low levels, don’t express well in bacteria due to loss of post-translational modifications, and are often unstable in detergent based purifications. Development of more methods to express, purify, and reconstitute lysosomal transporters into liposomes for biochemical characterization.

Key methods:

Comparison of protein overexpression: YP+Gal > YNB+Gal. Also pTEF> pGPD> pADH. pTEF and YP +Gal are comparable.
4 copies of Protease cleavable construct: pTEF1::Ypq1-HRV 3C protease site-GFP-8xHis cloned in integrative pRS305 and pRS306 backbones in both mating types. Generated diploids to maximize overexpression. Protein stability improved if using GFPS65T instead of yEGFP (yEGFP→GFPS65T: G65T, A72S, Q80R).
Cell lysis and purification: 4L YPD cultures, grown to high OD. Spheroplasted with zymolyase. Dounce homogenized. Solubilized proteins in 2% DDM. Affinity chromatography 8xHis with TALON cobalt resin. Cleavage with HRV 3C protease, followed by purification by size exclusion chromatography.
Purified Ypq1 in +Lysine conditions, when incubated in buffer- lysine, leads to instability and protein aggregation. Progressive reduction in lysine conc: Ypq1 stable in 10 mM and 1 mM, but not in 0.1 mM lysine. Reported lysine pools inside the vacuole (∼5-10 mM) and the cytoplasm (∼0.5-0.8 mM).
Reconstitution into proteoliposomes and transport: reconstituted Ypq1 into yeast polar lipid extract (Avanti Polar Lipids) in the presence of 10 mM lysine in neutral internal buffer (pH 7.2). Exchanged the proteoliposome into an external buffer containing 2 μM 14C-lysine and ATP.
Maximal uptake when intra proteoliposome pH is 6.2 and outside pH is 8.2 with a lysine gradient.

Major takeaways:

Massive overexpression does not result in constitutive degradation of Ypq1 in vivo.
Lysine and Arginine transport possible. Both can compete in the proteolipsosome based transport assay. But in vivo, removal of arginine does not lead to Ypq1 degradation.
PQ loop mutant (P229S,Q230R) resistant to degradation upon lysine withdrawal. Also stable in vitro +/- lysine. PQ mutant also defective in protein transport. PQ motif acts as a molecular hinge that allows PQ-loop transporters to change conformations during transport.