Molecular characterization of the heteromeric coenzyme A-synthesizing protein complex (CoA-SPC) in the yeastSaccharomyces cerevisiae

Abstract

Coenzyme A (CoA) as an essential cofactor for acyl and acetyl transfer reactions is synthesized in five enzymatic steps from pantothenate, cysteine, and ATP. In the yeast Saccharomyces cerevisiae, products of five essential genes CAB1-CAB5 (coenzyme A biosynthesis) are required to catalyze CoA biosynthesis. In addition, nonessential genes SIS2 and VHS3 similar to CAB3 are also involved. Using epitope-tagged variants of Cab3 and Cab5, we show that both proteins cofractionate upon chromatographic separation, forming a complex of about 330 kDa. We thus systematically investigated interactions among Cab proteins. Our results show that Cab2, Cab3, Cab4, and Cab5 indeed bind to each other, with Cab3 as the sole protein, which can interact with itself and other Cab proteins. Cab3 also binds to Sis2 and Vhs3 that were previously characterized as subunits of phosphopantothenoylcysteine decarboxylase. Pantothenate kinase encoded by CAB1 as the rate-limiting enzyme of CoA biosynthesis did not interact with other Cab proteins. Mapping studies revealed that the nonconserved N-terminus of Cab3 is required for dimerization and for binding of Cab2 and Cab5. Our interaction studies confirm early reports on the existence of a CoA-synthesizing protein complex (CoA-SPC) in yeast and provide precise data on protein domains involved in complex formation.

High confidence proteomic analysis of yeast LDs identifies additional droplet proteins and reveals connections to dolichol synthesis and sterol acetylation

“…Another protein, Nus1, has significant homology to Rer2 and Srt1, but does not have cis -IPTase activity (41). Both Srt1 (42) and Nus1 (22, 37, 43) have been localized previously to the LD. We found that Rer2 also localizes to the LD, as we detected Rer2-GFP in both membrane and LD fractions ( Fig.…”

“…The only de novo TG synthetic enzyme that we failed to detect in our LD proteome is Pah1, although it has previously been localized to the LD (36). Four of our six newly identified LD proteins increase the variety of lipid metabolic functions ascribable to the LD: Cab5 is involved in CoA synthesis (37), an important cofactor for many lipid synthesis reactions; Tsc10 catalyzes the second step in the synthesis of phytosphingosine, a long-chain base for sphingolipid production (38); Rer2 and Say1 function in dolichol and sterol metabolism, respectively, and are discussed below. Five proteins (Pet10, Yim1, YOR059C, and the newly identified YKL047W and YPR147C) do not have clear functions in yeast (see Table 1), but are likely to be involved in lipid metabolism as well.…”

“…Further, we show that two of these proteins, Say1 and Rer2, are lipid metabolic enzymes that are active in LD fractions. One of the proteins that we newly identified, Cab5, is involved in CoA biosynthesis with Cab2, Cab3, and Cab4 (37). Although none of Cab2–4 were identified as LD proteins in our proteome, by fluorescence microscopy, we were additionally able to detect Cab4-GFP at the LD (data not shown).…”

InhibitingMycobacterium tuberculosisCoaBC by targeting a new allosteric site

“…This fused arrangement might also help to channel the CoaB product to the CoaC active site more effectively. The human CoaB and other eukaryotic orthologues form stable dimers due to the extra dimerisation region (Figure S5), but is also known in yeast that the entire CoA pathway assembles into a metabolon centred on CoaC 35,36 (known as CAB3). This hints that close proximity between the different active sites of the CoA enzymes is desirable and that substrate channelling of products and substrates between different enzymes might be important in this pathway.…”

Coenzyme A and its derivatives: renaissance of a textbook classic

“…This is currently the subject of intensive research owing to the possibility of interactions with key signalling proteins, such as S6K (S6 kinase) and mTOR (mammalian target of rapamycin) [15]. A multifunctional CoA biosynthetic complex exists in yeast, with the Cab3 subunit of PPCD (phosphopanthenoylcysteine decarboxylase) acting as a scaffold for a CoA-synthesizing protein complex (CoA-SPC), which intersects with signalling pathways involved in salinity tolerance and cell cycle progression [16,17].…”