Identification of Putative Substrates for the Periplasmic Chaperone YfgM in Escherichia coli Using Quantitative Proteomics

Abstract

How proteins are trafficked, folded, and assembled into functional units in the cell envelope of Gram-negative bacteria is of significant interest. A number of chaperones have been identified, however, the molecular roles of these chaperones are often enigmatic because it has been challenging to assign substrates. Recently we discovered a novel periplasmic chaperone, called YfgM, which associates with PpiD and the SecYEG translocon and operates in a network that contains Skp and SurA. The aim of the study presented here was to identify putative substrates of YfgM. We reasoned that substrates would be incorrectly folded or trafficked when YfgM was absent from the cell, and thus more prone to proteolysis (the loss-of-function rationale). We therefore used a comparative proteomic approach to identify cell envelope proteins that were lower in abundance in a strain lacking yfgM, and strains lacking yfgM together with either skp or surA. Sixteen putative substrates were identified. The list contained nine inner membrane proteins (CusS, EvgS, MalF, OsmC, TdcB, TdcC, WrbA, YfhB, and YtfH) and seven periplasmic proteins (HdeA, HdeB, AnsB, Ggt, MalE, YcgK, and YnjE), but it did not include any lipoproteins or outer membrane proteins. Significantly, AnsB (an asparaginase) and HdeB (a protein involved in the acid stress response), were lower in abundance in all three strains lacking yfgM. For both genes, we ruled out the possibility that they were transcriptionally down-regulated, so it is highly likely that the corresponding proteins are misfolded/mistargeted and turned-over in the absence of YfgM. For HdeB we validated this conclusion in a pulse-chase experiment.

A multi-host approach to identify a transposon mutant of Pseudomonas aeruginosa LESB58 lacking full virulence

“…Genotyping [ 7 ] of the STM PALES_11731 mutant revealed that the mini-Tn5- tet transposon was inserted in the 3′ region of gene PALES_11731 ( yfgM ), 10 nucleotides before the end of the gene’s sequence (Additional file 1 : Figure S1). PALES_11731 codes for YfgM, an ancillary SecYEG translocon subunit [ 22 ] for which the exact function remains unclear. The insertion introduces a stop codon and the last two amino acids of the translated protein are missing (Additional file 1 : Figure S2).…”

“…The predicted structure of the protein suggests that the mutation does not clearly affect the YfgM function. In Escherichia coli , an inactivation of yfgM increases the bacteria sensitivity to acidity [ 22 ]. The STM PALES_11731 mutant resistance to acid stress was, however, the same as for the wild-type strain (Additional file 1 : Table S2), supporting the idea that YfgM is still functional.…”

An expression tag toolbox for microbial production of membrane bound plant cytochromes P450

“…The vector encodes a T7 promoter upstream of the gene, a TEV protease cleavage site at the 3 0 end of the gene followed by a GFP folding reporter and an octahistidine tag (Norholm et al, 2013). The N-terminal tags were chosen based on their topologies and expression profiles determined in other studies (Daley et al, 2005;Lee et al, 2013;Sletta et al, 2007), membrane localization in E. coli (Gotzke et al, 2015) or relaxation of mRNA folding energy (Kudla et al, 2009). The N-terminal tag-types fall in five categories ( Fig.…”

Conditional Proteolysis of the Membrane Protein YfgM by the FtsH Protease Depends on a Novel N-terminal Degron

“…Therefore, we tested whether the previously reported YfgM interactors PpiD (31)(32)(33)(34) and RcsB (35) influence proteolysis. Because the degradation profiles were not altered in the ⌬ppiD (Fig.…”

“…It may mediate the transport of proteins released from the SecYEG translocon to periplasmic chaperones like SurA or Skp (32,34). On the other hand, YfgM interacts with the cytoplasmic response regulator RcsB of the Rcs phosphorelay system (35).…”