Functional characterization of all‐trans retinoic acid‐induced differentiation factor (ATRAID)

All‐trans retinoic acid‐induced differentiation (ATRAID) factor was first identified in HL60 cells. Several mRNA isoforms exist, but the respective proteins have not been fully characterized. In transfected cells expressing Myc‐Flag‐tagged ATRAID Isoform (Iso) A, B, and C, Iso C was found to be expressed at high levels, Iso A was found to be expressed at low levels due to rapid degradation, and the predicted protein expressed from Iso B was not detected. Iso C was present mainly in an N‐glycosylated form. In subcellular fractionation experiments, Iso C localized to the membranous and nuclear fractions, while immunofluorescence analysis revealed that Iso C is located close to the plasma membrane, mainly in cytoplasmic vesicles and in the Golgi area. We confirm that Iso C colocalizes to some extent with endosomal/lysosomal markers LAMP1 and LAMP2. Furthermore, we show that ATRAID co‐localizes with RAB11, a GTPase associated with recycling endosomes and implicated in regulating vesicular trafficking. ATRAID isoform A and C are indicated. Iso C is N‐glycosylated and found in high densities close to the nucleus where it colocalizes with Golgi markers GALNT2 and GM130. Iso C colocalizes with proteins LAMP1, LAMP2, GALNT2, and GM130 as well as RAB11, a marker for recycling endosomes. Iso A is rapidly degraded by a proteasomal degradation pathway.