A Genome-Wide Enhancer Screen Implicates Sphingolipid Composition in Vacuolar ATPase Function in Saccharomyces cerevisiae

Abstract

The function of the vacuolar H 1 -ATPase (V-ATPase) enzyme complex is to acidify organelles; this process is critical for a variety of cellular processes and has implications in human disease. There are five accessory proteins that assist in assembly of the membrane portion of the complex, the V 0 domain. To identify additional elements that affect V-ATPase assembly, trafficking, or enzyme activity, we performed a genome-wide enhancer screen in the budding yeast Saccharomyces cerevisiae with two mutant assembly factor alleles, VMA21 with a dysfunctional ER retrieval motif (vma21QQ ) and vma21QQ in combination with voa1D, a nonessential assembly factor. These alleles serve as sensitized genetic backgrounds that have reduced V-ATPase enzyme activity. Genes were identified from a variety of cellular pathways including a large number of trafficking-related components; we characterized two redundant gene pairs, HPH1/ HPH2 and ORM1/ORM2. Both sets demonstrated synthetic growth defects in combination with the vma21QQ allele. A loss of either the HPH or ORM gene pairs alone did not result in a decrease in vacuolar acidification or defects in V-ATPase assembly. While the Hph proteins are not required for V-ATPase function, Orm1p and Orm2p are required for full V-ATPase enzyme function. Consistent with the documented role of the Orm proteins in sphingolipid regulation, we have found that inhibition of sphingolipid synthesis alleviates Orm-related growth defects.

Nitrate Reductase Knockout Uncouples Nitrate Transport from Nitrate Assimilation and Drives Repartitioning of Carbon Flux in a Model Pennate Diatom

“…Recently, similar sphingolipid desaturases have been characterized in the diatom T. pseudonana (Michaelson et al, 2013;Tonon et al, 2005). In yeast, and in Arabidopsis, regions within the vacuolar bilayer, enriched in sphingolipids and sterols (Chung et al, 2003;Finnigan et al, 2011), have been identified as essential to the function of vacuolar-ATPases (Ozolina et al, 2013;Yoshida et al, 2013). Vacuolar-ATPases are the proton pumps in plants (reviewed in Martinoia et al, 2007) that provide energy for NO 3 2 transport into the vacuole via NO 3 2 ClC transporters identified in this study.…”

Alteration of complex sphingolipid composition and its physiological significance in yeast Saccharomyces cerevisiae lacking vacuolar ATPase

“…For example, in S. cerevisiae, the proton-pump activity of V-ATPase decreases on deletion of ELO3 or ORM1 and ORM2, all of which are involved in sphingolipid biosynthesis (Chung et al, 2003;Finnigan et al, 2011). In addition, impaired biosynthesis of ergosterol also causes a reduction in V-ATPase activity (Zhang et al, 2010).…”

“…It was previously reported that deletion of ELO3 or ORM1 and ORM2 causes a reduction in the activity of V-ATPase (Chung et al, 2003;Finnigan et al, 2011). ELO3 is involved in synthesis of very long-chain fatty acids, which are incorporated into sphingolipids (Oh et al, 1997), and ORM1 and ORM2 encode negative regulators of serine palmitoyltransferase, which catalyses the initial step of de novo sphingolipid biosynthesis (Breslow et al, 2010), suggesting a functional connection between sphingolipids and V-ATPase.…”

Intracellular Proton Pumps as Targets in Chemotherapy: V-ATPases and Cancer

“…Despite V 1 being the cytoplasmic domain, C26-acyl group-containing sphingolipids are necessary to generate ATPase-competent V 1 domains [82]. Other studies have corroborated genetically the importance of sphingolipids for V-ATPase function [83]. Moreover, luminal acidification of melanocyte trans-Golgi system and endo/lysosomes have been suggested to depend on glycosphingolipids, since a cell line devoid of glucosylceramide synthase presents defective luminal acidification [84].…”