COMPARATIVE GENE ) is a key regulator of lipid metabolism and signaling in mammals, but its underlying mechanisms are unclear. Disruption of CGI-58 in either mammals or plants results in a significant increase in triacylglycerol (TAG), suggesting that CGI-58 activity is evolutionarily conserved. However, plants lack proteins that are important for CGI-58 activity in mammals. Here, we demonstrate that CGI-58 functions by interacting with the PEROXISOMAL ABC-TRANSPORTER1 (PXA1), a protein that transports a variety of substrates into peroxisomes for their subsequent metabolism by b-oxidation, including fatty acids and lipophilic hormone precursors of the jasmonate and auxin biosynthetic pathways. We also show that mutant cgi-58 plants display changes in jasmonate biosynthesis, auxin signaling, and lipid metabolism consistent with reduced PXA1 activity in planta and that, based on the double mutant cgi-58 pxa1, PXA1 is epistatic to CGI-58 in all of these processes. However, CGI-58 was not required for the PXA1-dependent breakdown of TAG in germinated seeds. Collectively, the results reveal that CGI-58 positively regulates many aspects of PXA1 activity in plants and that these two proteins function to coregulate lipid metabolism and signaling, particularly in nonseed vegetative tissues. Similarities and differences of CGI-58 activity in plants versus animals are discussed.

SEIPIN proteins are localized to endoplasmic reticulum (ER)-lipid droplet (LD) junctions where they mediate the directional formation of LDs into the cytoplasm in eukaryotic cells. Unlike in animal and yeast cells, which have single SEIPIN genes, plants have three distinct SEIPIN isoforms encoded by separate genes. The mechanism of SEIPIN action remains poorly understood, and here we demonstrate that part of the function of two SEIPIN isoforms in Arabidopsis (Arabidopsis thaliana), AtSEIPIN2 and AtSEIPIN3, may depend on their interaction with the vesicle-associated membrane protein (VAMP)-associated protein (VAP) family member AtVAP27-1. VAPs have well-established roles in the formation of membrane contact sites and lipid transfer between the ER and other organelles, and here, we used a combination of biochemical, cell biology, and genetics approaches to show that AtVAP27-1 interacts with the N termini of AtSEIPIN2 and AtSEIPIN3 and likely supports the normal formation of LDs. This insight indicates that the ER membrane tethering machinery in plant cells could play a role with select SEIPIN isoforms in LD biogenesis at the ER, and additional experimental evidence in Saccharomyces cerevisiae supports the possibility that this interaction may be important in other eukaryotic systems.

Cytoplasmic lipid droplets (LDs) are evolutionarily conserved organelles that store neutral lipids and play critical roles in plant growth, development and stress responses. However, the molecular mechanisms underlying their biogenesis at the endoplasmic reticulum (ER) remain obscure. Here we show that a recently identified protein termed LDIP (LD-Associated Protein [LDAP]-Interacting Protein) works together with both endoplasmic reticulum-localized SEIPIN and the LD-coat protein LDAP to facilitate LD formation in Arabidopsis thaliana. Heterologous expression in insect cells demonstrated that LDAP is required for the targeting of LDIP to the LD surface, and both proteins are required for the production of normal numbers and sizes of LDs in plant cells. LDIP also interacts with SEIPIN via a conserved hydrophobic helix in SEIPIN and LDIP functions together with SEIPIN to modulate LD numbers and sizes in plants. Further, co-expression of both proteins is required to restore normal LD production in SEIPIN-deficient yeast cells. These data, combined with the analogous function of LDIP to a mammalian protein called LD Assembly Factor 1, are discussed in the context of a new model for LD biogenesis in plant cells with evolutionary connections to LD biogenesis in other eukaryotes.