Profile of Membrane Cargo Trafficking Proteins and Transporters Expressed under N Source Derepressing Conditions in Aspergillus nidulans.

Sofia Dimou,Eleana Sarantidi,Xenia Georgiou,Athanasios K Anagnostopoulos,George Diallinas

doi:10.3390/jof7070560

Abstract

Solute and ion transporters are proteins essential for cell nutrition, detoxification, signaling, homeostasis and drug resistance. Being polytopic transmembrane proteins, they are co-translationally inserted and folded into the endoplasmic reticulum (ER) of eukaryotic cells and subsequently sorted to their final membrane destination via vesicular secretion. During their trafficking and in response to physiological/stress signals or prolonged activity, transporters undergo multiple quality control processes and regulated turnover. Consequently, transporters interact dynamically and transiently with multiple proteins. To further dissect the trafficking and turnover mechanisms underlying transporter subcellular biology, we herein describe a novel mass spectrometry-based proteomic protocol adapted to conditions allowing for maximal identification of proteins related to N source uptake in A. nidulans. Our analysis led to identification of 5690 proteins, which to our knowledge constitutes the largest protein dataset identified by omics-based approaches in Aspergilli. Importantly, we detected possibly all major proteins involved in basic cellular functions, giving particular emphasis to factors essential for membrane cargo trafficking and turnover. Our protocol is easily reproducible and highly efficient for unearthing the full A. nidulans proteome. The protein list delivered herein will form the basis for downstream systematic approaches and identification of protein–protein interactions in living fungal cells.

Highlights

Transporters are essential transmembrane proteins that mediate the selective translocation of nutrients, metabolites, ions or drugs across cell membranes
This work primarily aimed to survey the proteome profile of proteins expressed in young actively growing hyphae under conditions that favour the transcriptional expression of transporters of solutes than can serve as secondary N sources, such as nucleobases, ureides, amino acids, nitrate, nitrite and generally any nutrient that can be metabolized to ammonium [19,21]
Validation of the proteome obtained showcased that tentatively all proteins known or predicted to be essential for membrane cargo trafficking and conventional secretion in fungi were identified

Summary

Introduction

Transporters are essential transmembrane proteins that mediate the selective translocation of nutrients, metabolites, ions or drugs across cell membranes. Their function is related to cell nutrition, communication, homeostasis and response to drugs and stress. Their malfunction is associated with genetic or metabolic diseases and drug resistance [1,2]. Given their importance in sensing the environment and cell communication, transporter regulation of biogenesis and turnover is highly regulated at multiple levels. Considering that the genomes of eukaryotic cells are predicted to encode nearly 7–10% of transporters (http://www.membranetransport.org/transportDB2/index.html accessed on 20 May 2021), a rough estimate of the number of proteins involved in solute, ion and drug cellular transport might approach 20–25% of a genome’s translating capacity

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