In English

Investigation of the Cargo Recognition Step in Peroxisomal Import

Ye Lou
Göteborg : Chalmers tekniska högskola, 2011. 68 s.
[Examensarbete på avancerad nivå]

Peroxisomes are cellular organelles present in all eukaryotic cells, which contain enzymes that catalyze certain oxidative reactions and metabolic processes. Proteins are selectively imported across the membrane into the peroxisomal lumen in their folded and active form. Peroxin 5 (Pex5p) is a cycling receptor responsible for the translocation of matrix proteins equipped with a type 1 peroxisome targeting signal(PTS1). The PTS1 is usually a conserved C-terminal tripeptide (S/A/C–K/R/H–L/M)which interacts with the TPR domain of Pex5p.

In order to get a better understanding of the recognition step of the Pex5p import cycle, two model proteins were used: human alanine-glyoxylate aminotransferase (AGT) and human Peroxiredoxin 5 (PRDX5).

Although Pex5p-dependent, human AGT has a unique, non-consensus PTS1: - KKL. Six mutants (Q385A, P388A, K389A, K390A, K391A and K390A/K391A) were constructed, which are localized not only within the C-terminal tripeptide, but also in the immediate upstream region. According to the circular dichroism and enzyme activity measurements, the conformation of the mutants did not change and they were fully active when bound to Pex5p. The isothermal titration microcalorimetry (ITC)experiment of the K390A demonstrated a binding affinity 10 times higher than that of the wildtype AGT. And the ITC experiment as well as the GST-pull down assay revealed that the K391A mutant was unable to interact with Pex5p. The structure of K390A-Pex5p (TPR) complex was solved by X-ray crystallography, from which we noticed a significant shift of the Pex5p TPR domain’s position compared to the wildtype complex. The data indicates that the structural flexibility of the receptor molecule allows sufficient binding of cargoes with various PTS1s.

PRDX5 has a canonic PTS1 at its C-terminus: -SQL. However, there is no binding to the Pex5p receptor under laboratory conditions. To free up the PTS1, the N142A, Q161K and N142A/C47S/C151S/N157A mutants were constructed, and their interactions with Pex5p were tested by GST-pull down and ITC. As a result, Q161K was observed to interact with Pex5p, which was confirmed by analytical size exclusion chromatography coupled with static light scattering. An ITC dilution assay of Q161K revealed a critical transition concentration in the very low micromolar range. The rather abrupt transition indicates that the dimerization occurs only above the critical transition concentration when there are enough monomers in the solution to trigger it. It is also suggested that the monomer form of PRDX5 binds Pex5p.

Nyckelord: Cargo recognition, PTS1, Peroxisomal translocation, AGT, PRDX5

Publikationen registrerades 2011-10-20. Den ändrades senast 2013-04-04

CPL ID: 147480

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