ABSTRACT
INSIGs are proteins that underlie sterol regulation of the mammalian proteins SCAP (SREBP cleavage activating protein) and HMG-CoA reductase (HMGR). The INSIGs perform distinct tasks in the regulation of these effectors: they promote ER retention of SCAP, but ubiquitin-mediated degradation of HMGR. Two questions that arise from the discovery and study of INSIGs are: how do they perform these distinct tasks, and how general are the actions of INSIGs in biology? We now show that the yeast INSIG homologs NSG1 and NSG2 function to control the stability of yeast Hmg2p, the HMGR isozyme that undergoes regulated ubiquitination. Yeast Nsgs inhibit degradation of Hmg2p in a highly specific manner, by directly interacting with the sterol-sensing domain (SSD)-containing transmembrane region. Nsg1p functions naturally to limit degradation of Hmg2p when both proteins are at native levels, indicating a long-standing functional interplay between these two classes of proteins. One way to unify the known, disparate actions of INSIGs is to view them as known adaptations of a chaperone dedicated to SSD-containing client proteins.
Subject(s)
Hydroxymethylglutaryl CoA Reductases/metabolism , Membrane Proteins/metabolism , Molecular Chaperones/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Sterols/metabolism , Amino Acid Sequence , Animals , Anticholesteremic Agents/metabolism , Bridged Bicyclo Compounds, Heterocyclic/metabolism , Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)/genetics , Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)/metabolism , Humans , Hydroxymethylglutaryl CoA Reductases/genetics , Hydroxymethylglutaryl-CoA Reductase Inhibitors/metabolism , Intracellular Signaling Peptides and Proteins , Isoenzymes/genetics , Isoenzymes/metabolism , Lovastatin/metabolism , Membrane Proteins/genetics , Molecular Chaperones/genetics , Molecular Sequence Data , Promoter Regions, Genetic , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae Proteins/genetics , Sequence Alignment , Tricarboxylic Acids/metabolismABSTRACT
In humans and Saccharomyces cerevisiae the free glycosylphosphatidylinositol (GPI) lipid precursor contains several ethanolamine phosphate side chains, but these side chains had been found on the protein-bound GPI anchors only in humans, not yeast. Here we confirm that the ethanolamine phosphate side chain added by Mcd4p to the first mannose is a prerequisite for the addition of the third mannose to the GPI precursor lipid and demonstrate that, contrary to an earlier report, an ethanolamine phosphate can equally be found on the majority of yeast GPI protein anchors. Curiously, the stability of this substituent during preparation of anchors is much greater in gpi7Delta sec18 double mutants than in either single mutant or wild type cells, indicating that the lack of a substituent on the second mannose (caused by the deletion of GPI7) influences the stability of the one on the first mannose. The phosphodiester-linked substituent on the second mannose, probably a further ethanolamine phosphate, is added to GPI lipids by endoplasmic reticulum-derived microsomes in vitro but cannot be detected on GPI proteins of wild type cells and undergoes spontaneous hydrolysis in saline. Genetic manipulations to increase phosphatidylethanolamine levels in gpi7Delta cells by overexpression of PSD1 restore cell growth at 37 degrees C without restoring the addition of a substituent to Man2. The three putative ethanolamine-phosphate transferases Gpi13p, Gpi7p, and Mcd4p cannot replace each other even when overexpressed. Various models trying to explain how Gpi7p, a plasma membrane protein, directs the addition of ethanolamine phosphate to mannose 2 of the GPI core have been formulated and put to the test.
