Analysis of the co-translational assembly of the fungal fatty acid synthase (FAS)

Manuel Fischer,Martin Grininger,Barbara Mulinacci,Janet Vonck,Ronnald Vollrath,Peter Kötter,Mirko Joppe,Luciano Ciccarelli,Kosta Konstantinidis,Dieter Oesterhelt

doi:10.1038/s41598-020-57418-8

Abstract

The yeast fatty acid synthase (FAS) is a barrel-shaped 2.6 MDa complex. Upon barrel-formation, two multidomain subunits, each more than 200 kDa large, intertwine to form a heterododecameric complex that buries 170,000 Å2 of protein surface. In spite of the rich knowledge about yeast FAS in structure and function, its assembly remained elusive until recently, when co-translational interaction of the β-subunit with the nascent α-subunit was found to initiate assembly. Here, we characterize the co-translational assembly of yeast FAS at a molecular level. We show that the co-translationally formed interface is sensitive to subtle perturbations, so that the exchange of two amino acids located in the emerging interface can prevent assembly. On the other hand, assembly can also be initiated via the co-translational interaction of the subunits at other sites, which implies that this process is not strictly site or sequence specific. We further highlight additional steps in the biogenesis of yeast FAS, as the formation of a dimeric subunit that orchestrates complex formation and acts as platform for post-translational phosphopantetheinylation. The presented data supports the understanding of the recently discovered prevalence of eukaryotic complexes for co-translational assembly, and is valuable for further harnessing FAS in the biotechnological production of aliphatic compounds.

Highlights

The yeast fatty acid synthase (FAS) is a barrel-shaped 2.6 MDa complex
In order to analyze the co-translational assembly of subunits as an early step in yeast FAS assembly, we generated a set of mutants that modulate the interface of subunits in the co-translationally formed substructure
Earlier studies have shown that α is rapidly degraded in mutants lacking β20,21, so that the cytoplasm of yeast strains with assembly-defective FAS presumably contains only low levels of subunit α

Summary

Introduction

The yeast fatty acid synthase (FAS) is a barrel-shaped 2.6 MDa complex. Upon barrel-formation, two multidomain subunits, each more than 200 kDa large, intertwine to form a heterododecameric complex that buries 170,000 Å2 of protein surface. The architecture of fungal FAS was elucidated for the proteins from Saccharomyces cerevisiae (baker’s yeast)[5,6,7] and the thermophilic fungus Thermomyces lanuginosus[8], revealing an elaborate 2.6 MDa large α6β6 barrel-shaped complex that encapsulates fungal de novo FA synthesis in its interior (Fig. 1A). Notwithstanding a profound knowledge about this protein family, the biogenesis of fungal FAS has not been investigated until recently, when Shiber et al identified yeast FAS as initiating assembly via the co-translational interaction of subunits α (encoded by FAS2) and β (FAS1)[16]. Footprint data made perfectly sense from a structural perspective, because the C-terminus of β and the N-terminus of α intertwine (about 95 amino acids) to form the MPT domain (see Fig. 1A)

Methods

Results

Conclusion