Development of a novel biochemical assay for peroxisome biogenesis and cargo transport
Peroxisomes are membrane-bound organelles that support numerous biochemical functions, including breakdown of long-chain fatty acids and synthesis of lipids. Cells can produce peroxisomes through fission of existing peroxisomes, or build new peroxisomes de novo from vesicles that bud from the endoplasmic reticulum (ER). We report our efforts to develop a novel, luciferase-based biochemical assay of peroxisome biogenesis and cargo transport. Our assay involves an enzyme reporter, Gaussia luciferase (GLuc), split into two halves (GLucN & GLucC) that lack light-emitting activity on their own, but that assemble together into a functional enzyme. We fused GLucC to the luminal terminus of a peroxisome transmembrane protein (Pex3). When GLucC-Pex3 is transported from the ER to a peroxisome, the GLucC tag faces the lumen of the peroxisome. GLucN, was tagged at either the amino- or carboxy-termini with peroxisome import signals that allow GLucN to enter the peroxisome lumen and assemble with GLucC-Pex3. Control experiments in peroxisome-replete cells revealed all engineered and sequence-verified GLuc reporter constructs expressed in yeast and were detectable at expected molecular weight via Western blot. Unexpectedly, targeting soluble GLucN and GLucC to the peroxisome lumen yielded a 10-fold reduction in signal compared to cytosolic, split GLuc, indicating peroxisome lumen might be a non-permissive environment for GLuc. Fusing GLucC to the luminal terminus of Pex3 reduced signal ~10-fold further, a level barely above background. Phenotypic assays indicated the chimeric GLucC-Pex3 reporter failed to rescue the oleic acid growth defect of cells lacking Pex3, indicating that the GLucC tag disrupts function of Pex3.
"Development of a novel biochemical assay for peroxisome biogenesis and cargo transport,"
OSR Journal of Student Research: Vol. 5
, Article 295.
Available at: https://scholarworks.lib.csusb.edu/osr/vol5/iss1/295