Pnc1 piggy-back import into peroxisomes relies on Gpd1 homodimerisation

Abstract

Peroxisomes are eukaryotic organelles that posttranslationally import proteins via one of two conserved peroxisomal targeting signal (PTS1 or 2) mediated pathways. Oligomeric proteins can be imported via these pathways but evidence is accumulating that at least some PTS1-containing monomers enter peroxisomes before they assemble into oligomers. Some proteins lacking a PTS are imported by piggy-backing onto PTS-containing proteins. One of these proteins is the nicotinamidase Pnc1, that is co-imported with the PTS2-containing enzyme Glycerol-3-phosphate dehydrogenase 1, Gpd1. Here we show that Pnc1 co-import requires Gpd1 to form homodimers. A mutation that interferes with Gpd1 homodimerisation does not prevent Gpd1 import but prevents Pnc1 co-import. A suppressor mutation that restores Gpd1 homodimerisation also restores Pnc1 co-import. In line with this, Pnc1 interacts with Gpd1 in vivo only when Gpd1 can form dimers. Redirection of Gpd1 from the PTS2 import pathway to the PTS1 import pathway supports Gpd1 monomer import but not Gpd1 homodimer import and Pnc1 co-import. Our results support a model whereby Gpd1 may be imported as a monomer or a dimer but only the Gpd1 dimer facilitates co-transport of Pnc1 into peroxisomes.

Two NAD-linked redox shuttles maintain the peroxisomal redox balance in Saccharomyces cerevisiae

“…Furthermore, we have previously shown that replacing the active site residue lysine 245 with alanine, has a deleterious effect on activity. Although the mutant is stably expressed 18 and targeted to peroxisomes to a similar extent as wild type Gpd1p-GFP (Fig. 3D ), expression of Gpd1p K245A-GFP was unable to restore lysine biosynthesis in mdh3/gpd1Δ cells (Fig.…”

“…Our study emphasises a central role of peroxisomes in the regulation of NAD + metabolism. Gpd1p co-imports the nicotinamidase Pnc1p 13 , 18 , 28 . Its role inside peroxisomes is unclear as was the role of Gpd1p.…”

“…LYS1 and LYS12 were amplified by PCR containing 18 nucleotide flanking regions identical to each side of the intended insertion site and inserted into a Tpi1 promoter driven N-terminal expression vector derived from Ycplac33. All GPD1 expression constructs contained 523 bp upstream from the ATG as their promoter regions and have been described previously 18 . In brief, Gpd1p-mCherry-PTS1 was constructed by fusion of mCherry-SKL to the C-terminus of Gpd1p.…”

The budding yeast Pex5p receptor directs Fox2p and Cta1p into peroxisomes via its N-terminal region near the FxxxW domain

“…As a consequence, polypeptides with no peroxisomal targeting signal can enter peroxisomes while bound or 'piggy-backed' to polypeptides that contain one. This workaround mechanism was first demonstrated for thiolase (Glover et al, 1994), and evidence has since accumulated that this mechanism is widespread in S. cerevisiae (Yang et al, 2001;Effelsberg et al, 2015;Kumar et al, 2016;Saryi et al, 2017) and other species (Titorenko et al, 2002;Lee et al, 1997). Therefore, we tested whether the observed import of Fox2-GFP and Cta1p-GFP, which had their C-termini blocked with a GFP tag, might be due to the formation of complexes with the respective endogenous native proteins.…”

“…To date, only two proteins with functional PTS2 sequences have been identified in S. cerevisiae, 3-ketoacyl-CoA thiolase (Fox3p/Pot1p) (Erdmann, 1994) and glycerol-3-phosphate dehydrogenase (Gpd1p) (Jung et al, 2010). Curiously, the import of the nicotinamidase Pnc1p into peroxisomes also depends on the PTS2 signal, but it relies on the signal in Gpd1p via a piggy-back import mechanism (Effelsberg et al, 2015;Kumar et al, 2016;Saryi et al, 2017), another feature that is characteristic of peroxisome biogenesis.…”