Dataset Information

CryoEM architecture of a near-native stretch-sensitive membrane microdomain

ABSTRACT: Eisosomes, stress-sensitive membrane compartments in yeast, are scaffolded by the BAR-domain proteins Pil1 and Lsp1. We isolated near-native eisosomes as helical filaments of Pil1/Lsp1 lattice bound to plasma membrane lipids and solved their structure by helical reconstruction. From the same near-native eisosome samples, we performed mass spectroscopy to determine the protein content, and the abundance ratio of Pil1/Lsp1.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Nesli Ece Sen

LAB HEAD: Prof. Robbie Loewith

PROVIDER: PXD050326 | Pride | 2024-07-18

REPOSITORIES: Pride

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Cryo-EM architecture of a near-native stretch-sensitive membrane microdomain.

Kefauver Jennifer M JM Hakala Markku M Zou Luoming L Alba Josephine J Espadas Javier J Tettamanti Maria G MG Gajić Jelena J Gabus Caroline C Campomanes Pablo P Estrozi Leandro F LF Sen Nesli E NE Vanni Stefano S Roux Aurélien A Desfosses Ambroise A Loewith Robbie R

Nature 20240724 8025

Biological membranes are partitioned into functional zones termed membrane microdomains, which contain specific lipids and proteins<sup>1-3</sup>. The composition and organization of membrane microdomains remain controversial because few techniques are available that allow the visualization of lipids in situ without disrupting their native behaviour<sup>3,4</sup>. The yeast eisosome, composed of the BAR-domain proteins Pil1 and Lsp1 (hereafter, Pil1/Lsp1), scaffolds a membrane compartment that s ...[more]

PMID: 39048819

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Project description:Saccharomyces cerevisiae is an established microbial host for the production of non-native compounds. The synthesis of these compounds typically demands energy and competes with growth for carbon and energy substrate. Uncoupling product formation form growth would benefit product yields and decrease formation of by-product biomass. Studying non-growing metabolically-active yeast cultures provides a first step towards developing S. cerevisiae as a non-growing, robust cell factory. Non-growing metabolically-active cultures can be obtained in retentostat, a glucose-limited, continuous bioreactor system in which biomass accumulates while spent medium is constantly removed. Hitherto retentostat cultures of S. cerevisiae have only been reported under anaerobiosis, condition inappropriate for the production of energy-demanding products. The present study, using retentostat cultures, explores the physiology of non-dividing, fully respiring S. cerevisiae, focusing on industrially-relevant features. Following model-aided experimental design, retentostat cultivations were optimized for accelerated but smooth transition of S. cerevisiae from exponential growth to near-zero growth rates. During 20 days in retentostat the biomass concentration increased, leading very slow growth rates (specific growth rates below 0.001 h-1) but high culture viability (over 80% of viable cells). The maintenance requirement (mATP) was estimated at 0.64 mmolATP.gX-1.h-1, which is remarkably ca. 35% lower than the mATP measured in anaerobic retentostat cultures. Transcriptional down-regulation of genes involved in biosynthesis and up-regulation of stress-responsive genes towards near-zero growth rates corresponded well with data from anaerobic retentostats. More striking was the extreme heat-shock tolerance of S. cerevisiae, which exceeded by far previously reported heat shock tolerance of notoriously robust yeast cultures such as stationary phase cultures. Furthermore, while the metabolic fluxes in the retentostats were relatively low as a result of extreme caloric restriction, off-line measurements revealed that S. cerevisiae retained a high catabolic capacity. The high viability and extreme heat-shock tolerance revealed the robustness of S. cerevisiae at near-zero growth in retentostat. In addition, the relatively low maintenance requirements and high metabolic capacity under severe calorie restriction underline the potential of S. cerevisiae as a non-dividing microbial cell factory for the production of energy-intensive compounds. The retentostat is a promising tool to identify the molecular basis of this extreme robustness. The goal of the present study is to investigate the physiology of aerobic fully respiring S. cerevsiae at near-zero growth rates. Fundamental but industrially-relevant questions were addressed thanks to the design, implementation and study of aerobic retentostat cultivations enabling a rapid but smooth transition of S. cerevisiae from exponential growth to near-zero growth rates.

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