Fig 1: βFaar reduces LD‐ER apposition by binding RAB18, resulting in LD shrinkage. A) RNA pulldown assays were conducted using biotin‐labeled sense or antisense probes targeting βFaar, followed by mass spectrometry (MS) analysis of the retrieved RAB18 band. The corresponding peptide sequences are listed above the graphs. B) Immunoblotting analysis revealed representative images displaying the interaction between RAB18 and βFaar in the pulldown assay. C) Anti‐RAB18 RNA immunoprecipitation (RIP) was performed on 3T3‐L1 cell lysate transfected with si‐NC or si‐βFaar, and qRT‐PCR analysis revealed that precipitated RNAs contained RAB18–βFaar interaction, and si‐βFaar reduced the binding ability between RAB18 and βFaar. Gapdh was used as a control to validate this interaction. D) Mapping of RAB18‐binding domains of βFaar was carried out, including full‐length and truncated fragments of βFaar. Immunoblotting images showed RAB18 binding to different βFaar fragments in the RNA pulldown samples. E) Predicting βFaar‐RAB18 binding sites was done through catPARID databases (http://service.tartaglialab.com/). F,G) Co‐immunoprecipitation experiments with antibodies against RAB3GAP1 or RAB3GAP2 demonstrated that overexpression or knockdown of βFaar affected formal transformation of RAB18‐GTP in 3T3‐L1 cells transfected with oe/sg‐βFaar plasmid. H) Immunoblotting analysis revealed representative images displaying the binding site between RAB18 and βFaar in the pull‐down assay. I) Immunofluorescence of the interaction of RAB18 and LDs, as shown by representative images with a scale bar indicating 20 µm (n = 7 mice). J) Immunofluorescence of the interaction of ER and LDs, as shown by representative images with a scale bar indicating 20 µm (n = 7 mice). K) Representative electron microscopy (EM) images displayed the contact between ER and LDs in cells, indicated by red arrows pointing at ER cisternae structures (n = 6 mice). L) Representative coronal section MRI images and visceral and subcutaneous adipose tissue volume of HFD‐fed control, AAV‐oe‐Rab18, AAV‐oe‐βFaar and AAV‐oe‐Rab18 & AAV‐oe‐βFaar mice (n = 3 mice). M) Representative images depicted LDs labeled green in oe‐Rab18, oe‐Rab18‐MUT, oe‐Rab18+oe‐βFaar, or oe‐Rab18‐MUT+oe‐βFaar‐transfected 3T3‐L1 preadipocytes, confirming their presence under different experimental conditions, with a scale bar indicating 20 µm. N) Schematic diagram showing the mechanism by which βFaar promotes RAB18‐GTP transform to RAB18‐GDP, inhibiting LDs mature. Schematic illustration was drawn by figdraw. The fold change in mRNA expression was calculated via the 2−ΔΔCt method. The data are presented as the means ± SEMs. The p values obtained using a two‐tailed unpaired Student's t‐test or two‐way ANOVA are indicated; ***p < 0.001.