Genetic and proteomic evidences support the localization of yeast enolase in the cell surface

Abstract

Although enolase, other glycolytic enzymes, and a variety of cytoplasmic proteins lacking an N-terminal secretion signal have been widely described as located at the cell surface in yeast and in mammalian cells, their presence in this external location is still controversial. Here, we report that different experimental approaches (genetics, cellular biology and proteomics) show that yeast enolase can reach the cell surface and describe the protein regions involved in its cell surface targeting. Hybrid enolase truncates, fused at their C terminus with the yeast internal invertase or green fluorescent protein (GFP) as reporter proteins, proved that the 169 N-terminal amino acids are sufficient to target the protein to the cell surface. Furthermore, the enolase-GFP fusion co-localized with a plasma membrane marker. Enolase was also identified among membrane proteins obtained by a purification protocol that includes sodium carbonate to prevent cytoplasmic contamination. These proteins were analyzed by SDS-PAGE, trypsin digestion and LC-MS/MS for peptide identification. Elongation factors, mitochondrial membrane proteins and a mannosyltransferase involved in cell wall mannan biosynthesis were also identified in this fraction.

Deglycosylation systematically improves N-glycoprotein identification in liquid chromatography–tandem mass spectrometry proteomics for analysis of cell wall stress responses in Saccharomyces cerevisiae lacking Alg3p

“…This analysis robustly identified 26 proteins, with 19 identified after both AspN and trypsin digest, four identified only by trypsin and three only by AspN (Supplementary Table S1, Supplementary material). The proteins identified were consistent 8 with previous proteomic analyses of yeast cell wall fractions [28,[32][33][34]. In addition, AspN digest allowed identification of Egt2p (Supplementary Table S1, Supplementary material), which was not identified in these previous studies.…”

Homocysteine Methyltransferases Mht1 and Sam4 Prevent the Accumulation of Age-damaged (R,S)-AdoMet in the Yeast Saccharomyces cerevisiae

“…The finding that (R,S)-AdoMet accumulates when both the SAM4 and MHT1 genes are deleted suggests that these are the major enzymes involved in limiting its buildup. It thus appears that YMR321C, encoding a protein that is 99% identical to Sam4 over the last 103 amino acids, may not be involved in (R,S)-AdoMet metabolism (31)(32)(33). These results also suggest that (R,S)-AdoMet may not be simply excreted, or converted back to the S,S form via radical SAM enzymes, or spontaneously degraded.…”

Cytosolic Proteins Contribute to Surface Plasminogen Recruitment ofNeisseria meningitidis

“…amino acids of enolase are sufficient for secretion of the protein (36). Furthermore, no proteolytic cleavage seems to be involved in the secretion process, except perhaps the removal of the N-terminal methionine (36,54).…”

“…Even though no signal peptide for secretion or localization to the outer membrane has yet been identified, enolases of bacteria and eukaryotic cells have been shown to be localized in the outer membrane (11,21,43,56,60). Indeed, the recruitment of plasminogen to cellular surfaces by enolase seems to be a conserved pattern in different cells, including bacteria, fungi, and eukaryotic cells (5,36,38,41,46). A recent proteomic analysis identified enolase and DnaK as components of outer membrane vesicles derived from serogroup B N. meningitidis (24).…”