Role of Pex21p for Piggyback Import of Gpd1p and Pnc1p into Peroxisomes of Saccharomyces cerevisiae

(Fig. 1D, upper panel).Similar to pnc1∆ mutant (Fig. 1B), both nat3∆and mdm20∆ released mostly NAM not NA (Fig.1D, lower panel). Fig. 1E (Fig. 2E, top panel) had a lesser effect (Fig. 2E, bottom (Fig. 3B). Next, we tested whether deleting ATG14 was sufficient to restore NAD + levels in the NatB mutants. As shown in Fig. 3C (left panel), deleting ATG14did not rescue the low NAD + levels in nat3∆ cells. Similarly, deleting other factors contributing to NR and NAM production ( Fig. 1A and Fig. 2E) in nat3∆ cells also failed to restore the NAD + level back to wild type ( shown to restore actin cable formation in NatB mutants (23,24,28,29). Interestingly, both TPM1-oe and TPM1-5 largely blocked NA/NAM release in the nat3∆ mutant (Fig. 3D) 1A). N-terminal acetyltransferases have specific targets that are largely determined by the sequence of the first two amino acids. NatB acetylates proteins with a MET-retained residue, followed by ASP, GLU, GLN, or ASN as the second residue (25,26,39,40). Nma1 and Nma2 have a MET-ASP N-terminus, but neither protein has been identified as a NatB target.Similar to nat3∆ mutant, nma1∆ cells showed decreased NAD + levels, which could not be rescued by supplementing NR (Fig. 4B). As for the controls, NR efficiently restored the NAD + level in the npt1∆ mutant, which has functional NR salvage (Fig. 4B). The similarities between nma1∆ and nat3∆ mutants suggested that Nma1and Nma2 activities are likely reduced in nat3∆ cells. Since N-terminal acetylation may affect the turnover of target proteins, we first determined whether reduced Nma1/Nma2 activities were due to decreased Nma1/Nma2 protein levels. As shown in Fig. 4C, Nma1 and Nma2 protein levels were indeed decreased in nat3∆ cells (Fig. 4C). In addition to Nma1 and Nma2, NAD + homeostasis factors Bna2, Bna5and Hst1 are also potential NatB targets. Bna2and Bna5 are biosynthesis enzymes in the de novo pathway (Fig. 1A) DISCUSSIONIn this study we characterized NatB complex as a novel NAD + homeostasis factor.Mutants lacking components of the NatB complex, NAT3 and MDM20, produce and release excess amount of NA and NAM. Our studies showed two pathways downstream of NatB contribute to NAD + homeostasis (Fig. 5).In one, NatB is important for proper NAD + biosynthesis by regulating Nma1/Nma2. NatB mutants have low NAD + levels (Fig. 2C), and all NAD + precursors examined failed to restore the NAD + levels ( Fig. 4A and 4B). This suggests that a NAD + biosynthesis factor(s) required for utilization of all NAD + precursors is defected in NatB mutants. Nma1 and Nma2 are such targets because they are the only factors required for all three NAD + biosynthesis pathways (de novo, NA/NAM salvage, and NR salvage) (Fig. 1A), and the N-terminal amino acid sequences are a match for NatB acetylation. In addition, nma1∆and nat3∆ mutants showed similar NAD + utilization defects (Fig. 4B). Although Nma2 is present in both mutants, Nma2 is known to play a minor role in NAD + metabolism. Supporting this model, decreased Nma1 protein level was observed in t...

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“…In response to nutrient starvation and stress, Pnc1 expression is induced and becomes ubiquitous (15,59 Table 1). …”

Section: Discussionmentioning

confidence: 99%

A functional link between NAD+ homeostasis and N-terminal protein acetylation in Saccharomyces cerevisiae

Croft¹,

Raj²,

Salemi

et al. 2018

Journal of Biological Chemistry

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“…These proteins may be localized by internally located PTS peptides, by alternative targeting signals or pathways, by "piggy-backing" or by peripheral and integral membrane proteins [44,45]. For example, a key enzyme of the NAD + salvage pathway, Pnc1p, a nicotinamidase with no functional terminal PTS, is co-imported into peroxisomes by piggyback transport via the glycerol-producing PTS2 protein glycerol-3-phosphate dehydrogenase Gpd1p [46]. Further research is needed to determine how OsAld-Y is transported.…”

Section: Discussionmentioning

confidence: 99%

Identification of a peroxisomal-targeted aldolase involved in chlorophyll biosynthesis and sugar metabolism in rice

Zhang

Wang

et al. 2016

Plant Science

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“…For SOD1 it was shown that one of its chaperones, copper chaperone of SOD1 (CCS), does possess a PTS1 whereas for LDH it was recently found that translational readthrough of the stop codon in the LDHB transcript yields an extended isoform of this protein, the socalled LDHBx, which possesses a PTS1 and can form heterotetramers with both LDHA and LDHB [85,86]. Likewise, yeast Pnc1p was recently shown to interact with the PTS2-containing Gpd1p, a glycerol-3-phosphate dehydrogenase displaying a partial peroxisomal localization [87]. Apparently, all these PTS-less proteins reach the peroxisome matrix piggy-backed with the partners that do have one.…”

Section: Monomeric Vs Oligomeric Protein Import Modelmentioning

confidence: 98%

“…There are, however, three cases where import of oligomers occurs under conditions where there are no reasons to believe that PEX5 or other components of PIM are stoichiometrically limiting. One regards rat liver Cu/Zn superoxide dismutase 1 (SOD1) [85]; another, the two isoforms of human lactate dehydrogenase (LDH), LDHA and LDHB [86]; the third, the yeast nicotinamidase Pnc1p [87]. Indeed, none of these proteins possess a PTS.…”

Section: Monomeric Vs Oligomeric Protein Import Modelmentioning

confidence: 98%

The first minutes in the life of a peroxisomal matrix protein

Dias

Francisco

Rodrigues

et al. 2016

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research

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In the field of intracellular protein sorting, peroxisomes are most famous by their capacity to import oligomeric proteins. The data supporting this remarkable property are abundant and, understandably, have inspired a variety of hypothetical models on how newly synthesized (cytosolic) proteins reach the peroxisome matrix. However, there is also accumulating evidence suggesting that many peroxisomal oligomeric proteins actually arrive at the peroxisome still as monomers. In support of this idea, recent data suggest that PEX5, the shuttling receptor for peroxisomal matrix proteins, is also a chaperone/holdase, binding newly synthesized peroxisomal proteins in the cytosol and blocking their oligomerization. Here we review the data behind these two different perspectives and discuss their mechanistic implications on this protein sorting pathway.

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Role of Pex21p for Piggyback Import of Gpd1p and Pnc1p into Peroxisomes of Saccharomyces cerevisiae

Cited by 47 publications

References 57 publications

A functional link between NAD+ homeostasis and N-terminal protein acetylation in Saccharomyces cerevisiae

A functional link between NAD+ homeostasis and N-terminal protein acetylation in Saccharomyces cerevisiae

Identification of a peroxisomal-targeted aldolase involved in chlorophyll biosynthesis and sugar metabolism in rice

The first minutes in the life of a peroxisomal matrix protein

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