Distinct functions of acyl/alkyl dihydroxyacetonephosphate reductase in peroxisomes and endoplasmic reticulum

Honsho M, Tanaka M, Zoeller RA, and Fujiki Y. (2020) Distinct functions of acyl/alkyl dihydroxyacetonephosphate reductase in peroxisomes and endoplasmic reticulum. Frontiers in Cell and Developmental Biology

Plasmalogens are a class of phospholipids that contain a vinyl-ether bond at sn-1, which causes a more compact conformation of the lipid and results in unique characteristics. Of all the phospholipids in the human body, plasmalogens constitute almost 20% and this can range from 5-20% across different tissues. Plasmalogen biosynthesis occurs in the peroxisome and the endoplasmic reticulum (ER). Previous work has shown that the first two steps occur within the peroxisome and the final steps to produce plasmalogens are in the ER, but the location of the third enzyme in this pathway, acyl/alkyl DHAP reductase (ADHAPR), has not been clear. Previous work using a Chinese hamster ovary cell line FAA.K1B has suggested that ADHAPR may be localized to both the peroxisome and the ER. There is evidence to support another pathway that bypasses the step catalyzed by ADHAPR, since cells with a severe reduction in ADHAPR activity only showed a moderate plasmalogen reduction. If the product of the first two steps of the pathway cannot be reduced by ADHAPR, preventing the final four steps in plasmalogen synthesis to occur, it would be assumed that a strong decrease in plasmalogen levels would be detected. Other work using mouse embryonic cells showed a similar response when knocking down DHRS7b, a mammalian homolog for the yeast enzyme that catalyzes the reduction of 1-acyl-glycerone 3-phosphate. This work suggested that DHRS7b encodes the mammalian ADHAPR and Honsho et al sought to determine if the loss of ADHAPR activity has an influence on plasmalogen levels, as well as its subcellular localization.

To confirm whether the knockdown of DHRS7b reduces the production of plasmalogens, HeLa cells, a human cell line derived from cervical cancer cells, were transfected with two independent small interfering RNAs (siRNAs) against DHRS7b. It was found that transcription of ADHAPR was reduced by 60% in these cells compared to mock-treated cells. Further, plasmalogen ethanolamine (PlsEtn) and phosphotidylethanolamine (PtdEtn) were reduced by 40%. These findings confirm that ADHAPR encoded by DHRS7b has a role in plasmalogen biosynthesis and appears to catalyze the reduction of 1-O-alkyl-glycerone-3-phosphate.

Plasmalogen biosynthesis occurs in the peroxisome and ER, therefore Honsho et al wanted to determine where ADHAPR is localized in HeLa cells. Immunostaining with an anti-ADHAPR antibody found that it was co-localized with both the peroxisomal membrane protein, peroxin 14 (Pex14p) and calnexin, an ER molecular chaperone. In addition, immunostaining for FLAG-tagged ADHAPR (FLAG-ADHAPR) was also co-localized with both Pex14p and an ER marker protein Sec61β (EGFP-Sec61βC). Together, these findings indicate that ADHAPR localizes to both the ER and the peroxisome.

As ADHAPR is a type I integral membrane protein, it was theorized that the Pex19p-dependent class I pathway would be responsible for transporting this enzyme to the peroxisome. When HA2-Pex19pΔN23, a mutant cell line lacking 23 amino acids at the N-terminus which is essential for binding to Pex3p, immunofluorescence showed that FLAG-ADHAPR was synthesized, but unable to be delivered to the peroxisome. When Pex3p-depleted cells were used in import assays the transport of FLAG-ADHAPR was also inhibited. These findings indicate that for ADHAPR to target the peroxisome in HeLa cells, this is accomplished through a Pex19p- and Pex3p-dependent class I pathway.

Although they were able to provide evidence for the transport pathway to the peroxisome, the method of targeting to the ER is still unknown. In cells lacking Pex19p or Pex3p, FLAG-ADHAPR was still localized to the ER, indicating that the mechanism involved is independent of the Pex19p-Pex3p-dependent class I pathway. Next, the signal recognition particle (SRP) pathway, an ER targeting mechanism mediated by a N-terminal hydrophobic signal sequence was investigated. To determine if ADHAPR is co-translationally imported into the ER through the SRP pathway, the protein levels of FLAG-ADHAPR in SRP54-depleted HeLa cells was analyzed. The SRP54-depleted cells showed a reduction by more than half compared to the mock-treated cells, suggesting that the SRP pathway is involved in the transport of ADHAPR into the ER.

Honsho et al sought to confirm whether DHRS7b encodes ADHAPR, determine the role of ADHAPR in PlsEtn and PtdEtn biosynthesis, and identify where this enzyme localizes in HeLa cells. ADHAPR is the third enzyme in plasmalogen synthesis and was found to localize to both peroxisomes and the ER through independent pathways. Whether any functional differences of ADHAPR are present when localized to different organelles has not been eludicated. Some work has been done to suggest that the third step in plasmalogen synthesis occurs from ER-targeted ADHAPR. Honsho et al hypothesize that localization of the enzyme may be tissue specific as ADHAPR is highly expressed in a variety of tissues including liver, white adipocytes and the brain and these different tissues may require different lipids to be synthesized. As we increase our knowledge of the biosynthetic pathway of plasmalogens, including the enzymes and proteins involved, we will be able to advance our diagnostic and therapeutic approach towards diseases associated with a plasmalogen reduction.