Arabidopsis TMPIT/TMEM120/NET29 homologs are required for cell survival, Golgi morphology, and metabolic fluxes of very long-chain fatty acids to waxes and sphingolipids

The TMPIT/TMEM120/NET29 family of transmembrane proteins is conserved in eukaryotes, but its functions are controversial in mammals and have not been reported in plants. Structurally, these proteins were supposed to contain a coenzyme A (CoA)-binding motif and exhibit high similarity to that of ELOVL7, an enzyme involved in VLCFA (very long-chain fatty acid) elongation. Here, we report that due to functional redundancy of Arabidopsis TMPIT1 and TMPIT2 , single null-type mutants grew normally. However, they constitute an essential gene pair for cell survival after the first meiotic division; thus, only double heterozygotes could be maintained in the laboratory. The leakage-type double mutants, where tmpit1 is leaky and tmpit2 is null, are viable with a visible defect of epicuticular waxes-derivatives of VLCFAs. Wild-type TMPIT1 and TMPIT2 were targeted to the cis -Golgi network, endoplasmic reticulum, and nuclear envelope. The leaky mutations, carrying short deletions in the N-terminus cytosolic region of TMPIT1, blocked the Golgi localization, leading to abnormal Golgi morphology. Free VLCFA levels in these mutants were increased, but C24/C26 sphingolipid levels—another group of VLCFA derivatives—were reduced. Detailed complementary tests revealed that TMPITs from barley, tomato, and rubber tree rescued the wax phenotypes of Arabidopsis mutants. ELOVL7, even after adding the N-terminus of Arabidopsis TMPIT1, could not rescue Arabidopsis mutants, indicating that the mechanism of TMPITs and ELOVL7 differ. A point mutation that destroys the CoA binding motif of Arabidopsis TMPIT1 simultaneously damages its functionality. Our work provides genetic evidence that plant TMPITs regulate the metabolic fluxes of VLCFAs to waxes and sphingolipids. • Arabidopsis TMPIT1 and TMPIT2 constitute an essential gene pair for cell survival. • The leaky mutation on TMPIT1 block Golgi localization and affect Golgi morphology. • Plant TMPITs are new genes for metabolic fluxes of VLCFAs to waxes and sphingolipids.