The ubiquitin-like (UBX)-domain-containing protein Ubx2/ Ubxd8 regulates lipid droplet homeostasis

Abstract

SummaryLipid droplets (LDs) are central organelles for maintaining lipid homeostasis. However, how cells control the size and number of LDs remains largely unknown. Herein, we report that Ubx2, a UBX-domain-containing protein involved in endoplasmic reticulum (ER)-associated degradation, is crucial for LD maintenance. Ubx2 redistributes from the ER to LDs when LDs start to form and enlarge during diauxic shift and in the stationary phase. ubx2D cells contain abnormal numbers of LDs that are smaller than normal, and their triacylglycerol (TAG) is reduced to 50% of the normal level. Deletion of either the UBX or UBA domain in Ubx2 has no effect, but deletion of both causes LD phenotypes similar to that in ubx2D. The reduced level of TAG in ubx2D is probably the result of mislocalization of phospholipid:diacylglycerol acyltransferase (Lro1), one of the two TAG-synthesizing enzymes in yeast, which moves along the ER and distributes dynamically to the putative LD assembly sites abutting LDs. Thus, Ubx2 is important for the maintenance of cellular TAG homeostasis probably through Lro1. The mammalian Ubxd8 (also known as FAF2), when expressed in yeast, complements the defect of ubx2D, implying a functional conservation for these UBX-domain-containing proteins in lipid homeostasis.

Size control of lipid droplets in budding yeast requires a collaboration of Fld1 and Ldb16

“…Cells were imaged by taking 10 optical section images spaced at 0.5 mm captured by the Olympus IX81 fluorescence microscope using the green fluorescent protein (GFP) filter (excitation HQ470/40 nm, emission HQ525/50 nm) with a 1006 objective lens with numerical aperture (NA) 1.4 and a CoolSNAP TM HQ 2 CCD camera (Photometrics). We used the analysis LS professional software (Olympus) for image acquisition and processing, and Metamorph (Molecular Devices) for LD quantification, as described previously (Wang and Lee, 2012). The dual-or tri-color images were acquired by a DeltaVision system (Applied Precision) with 1006 objective lens (NA51.4) and a CoolSNAP TM HQ 2 charge-coupled device (CCD) camera (Photometrics) controlled by softWorx Suite (Applied Precision).…”

“…We used a freeze-substitution method, as described previously, for sample preparation (Wang and Lee, 2012). Ultrathin sections of 70-90 nm were placed on copper grids and stained with uranyl acetate and lead citrate.…”

“…Antibodies against Erg1, Erg6, Pet10 and Ubx2 have been described previously (Wang and Lee, 2012). Antibodies against Kar2, CPY, Sec61 and Pgk1 were gifted by the Schekman lab (UC Berkeley, Berkeley, CA), an antibody against protein A was obtained from Jackson ImmunoResearch, an antibody against Myc was obtained from Covance and antibodies against actin or ubiquitin were obtained from Millipore.…”

Molecular Mechanisms and Physiological Significance of Organelle Interactions and Cooperation

“…Experiments are lacking to probe for barriers to lipid trafficking at ER/droplet junctions or the extent to which new lipid synthesis is concentrated at junctions. The yeast diacylglycerol acyltransferase Lro1p is localized to ER/droplet junctions, suggesting that synthesis from this source is indeed coupled to droplet expansion (Wang and Lee, 2012).…”

“…Conversely, trafficking of UBXD8 to droplets resulted in an increase in neutral lipid due to inhibition of the triglyceride lipase, ATGL, by promoting dissociation of its activator CGI-58 (Olzmann et al, 2013). Similarly, deletion of UBX2 (yeast UBXD8) resulted in reduced levels of triacylglycerol (Wang and Lee, 2012).…”