Discovery of a Regulatory Subunit of the Yeast Fatty Acid Synthase

Abstract

Highlights d Discovery of a regulatory g subunit of the yeast fatty acid synthase (FAS) d The g subunit regulates FAS reductase activities by kinetic hysteresis d Establishes structural basis for g subunit-mediated FAS activity regulation d The g subunit defines a higher efficiency FAS functional compartment

Current limitations to high-resolution structure determination by single-particle cryoEM

“…(a) Amorphous carbon films have been the standard support for biological electron microscopy at room temperature since the early 1950s (Bradley, 1954), but they have also been used for high-resolution cryoEM of soluble (Bai et al ., 2013; Nguyen et al ., 2015; Haselbach et al ., 2018; Jahagirdar et al ., 2020) and membrane protein complexes (Schraidt and Marlovits, 2011; D'Imprima et al ., 2017; van Pee et al ., 2017; Menny et al ., 2018; Singh et al ., 2020). Amorphous carbon films are inexpensive and easily prepared in the EM laboratory (Booth et al ., 2011).…”

Structural insight intoPichia pastorisfatty acid synthase

“…While our understanding of yeast FAS structure is growing, our library of structures remains incomplete. The vast majority of published solution structures of FAS are from S. cerevisiae 13,[16][17][18][19] . To date, no structures are available for Pichia pastoris FAS from either cryoEM or X-ray crystallography.…”

Unstructured regions of large enzymatic complexes control the availability of metabolites with signaling functions

“…It was previously thought that this cycle was not regulated by FAS itself, and that the binding of ACP to various sites was stochastic and asymmetric, as indicated by molecular dynamics simulation studies [ 2 ]. The recent discovery of the γ subunit in yeast FAS showed that FAS enzymatic activity is regulated by the γ subunit in response to an abundance of its cosubstrate NADP and has impacts on the higher-order structure and consequently the conformational space probed by ACP [ 72 ]. ACP (residues 2125–2192) is tethered to flexible regions on either side of its termini (residues 2066–2124 at the N-ter, and residues 2125–2241 at the C-ter), which have not yet been captured by crystallography or electron cryomicroscopy at high resolution.…”

“…ACP (residues 2125–2192) is tethered to flexible regions on either side of its termini (residues 2066–2124 at the N-ter, and residues 2125–2241 at the C-ter), which have not yet been captured by crystallography or electron cryomicroscopy at high resolution. Additionally, mobile ACP is not frequently captured in the context of larger FAS structures [ 35 , 38 , 72 ], which points to a significant mobility caused by the connecting flexible regions. These flexible linkers are also predicted to include disordered regions and have only been seen at low resolution with cryo-EM [ 20 ].…”

“…2 ) , tethered to flexible regions with the aim of bringing the substrate from one enzymatic site to the other, it is reasonable to assume that the common feature of the manifested disorder observed in these complexes may contribute to the conformational space that is being explored by the flexible arms. Human and fungal FAS have two and six lipoyl arms, respectively, which are spatially confined, but can intercommunicate within their respective spatial constraints as defined by the length of the tethered flexible linker regions [ 44 ] in combination with the suppression of futile catalytic cycles by the γ subunit [ 72 ]. The mechanisms of the higher-order regulation of multiple carrier proteins is also currently unknown, since they must be spatially confined in order to avoid unintended protein interactions; in the case of FAS in yeast or C. thermophilum , this is succeeded by a chamber-like structure [ 35 , 42 ], but in the case of its human equivalent, the identity of the regulation mechanisms remains an open question because of the absence of resolved native FAS complexes.…”