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

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.

Comment on Dimou et al. Profile of Membrane Cargo Trafficking Proteins and Transporters Expressed under N Source Derepressing Conditions in Aspergillus nidulans. J. Fungi 2021, 7, 560

“…Evidence against AN11127 being an isofunctional Sec12 orthologue comes from the observation that is has no amino acid similarity with Sec12 proteins, and importantly, is not present in several Aspergilli and most ascomycetes." Dimou et al [9] cast doubts on our report on the identification of the gene encoding Sec12 in the filamentous fungal model A. nidulans as the product of AN11127, which might mislead readers to believe that our conclusions were wrong. We shall therefore summarize experimental evidence that overwhelmingly supports our conclusion that the product of AN11127 is indeed Sec12.…”

“…This same fusion protein labelled the ER intensely when expressed under the control of the moderately strong gpdA m promoter [8], strongly indicating that the AN11127 protein present in very low levels when its synthesis is driven by its own promoter. These low levels expression possibly contributed to the inability of Dimou et al [9] to detect the protein in their analyses.…”

“…This became apparent when trying to identify the S. cerevisiae Sec12 using the H. sapiens sequence; multiple rounds of psi-BLAST were required to show that they are indeed homologs, as BLASTp did not provide enough sensitivity to do so". This obstacle possibly contributed to the inability of Dimou et al [9] to identify sequence similarities between Sec12p and AN11127.…”

“…AN11127 has no amino acid similarity with Sec12 proteins, and importantly, is not present in several Aspergilli and most ascomycetes [9].…”

“…On July 2021, the Journal of Fungi published a paper titled 'Profile of Membrane Cargo Trafficking Proteins and Transporters Expressed under N Source Derepressing Conditions in Aspergillus nidulans', by Sofia Dimou et al [9]. On page 11 of this paper these authors discuss their failure to detect Sec12 among the catalogue of secretory proteins analyzed in their proteomic work with the following sentences: "Impressively (sic erat scriptum), all proteins essential for cargo biogenesis were indeed present in the proteome analyzed.…”