The effect of carbon source on the secretome of Kluyveromyces lactis

ArticleinProteomics 9(20):4744-54 · October 2009with12 Reads
Impact Factor: 3.81 · DOI: 10.1002/pmic.200800915 · Source: PubMed
Abstract

A proteomic analysis was performed on spent fermentation medium following bioreactor propagation of a wild-type industrial strain to identify proteins naturally secreted by Kluyveromyces lactis cells. Here, we report changes detected in the K. lactis secretome as a result of growth in three different carbon sources: glucose, galactose and glycerol. A total of 151 secreted proteins were detected by multi-dimensional separations and reversed-phase online nanoESI-MS/MS analysis. From these, we were able to identify 63 proteins (termed the "base secretome") that were common to all three fermentation conditions. The majority of base secretome proteins, 79%, possessed general secretory pathway (GSP) sequences and were involved with cell wall structure, glycosylation, carbohydrate metabolism and proteolysis. There was little variation in the functional groupings of base secretome GSP proteins and GSP proteins that were not part of the base secretome. In contrast, the majority of non-GSP proteins detected were not part of the base secretome and the functions of these proteins varied significantly. Finally, through further identification of non-GSP proteins in carbon sources not originally tested, we have gained further evidence of a protein export mechanism separate from the GSP in K. lactis.

    • "In the classical pathway, proteins that contain a specific N-terminal signal sequence are translocated into the ER, transported to the Golgi and then secreted by secretory vesicles [30,32,33]. Accumulating evidence indicates that a large number of signal-less proteins can also be secreted through the non-classical pathway [1,2,4,5,10,21,23,31], [34], including metabolic enzymes, chaperones, translation factors, and transcriptional regulators [5,8,9,11,20-24,31,34-37]. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a glycolytic enzyme and has been shown by immuno-transmission electron microscopy (immuno-TEM) to be present on the surface of Candida albicans and Saccharomyces cerevisiae[14]. "
    [Show abstract] [Hide abstract] ABSTRACT: Protein secretion is a fundamental process in all living cells. Proteins can either be secreted via the classical or non-classical pathways. In Saccharomyces cerevisiae, gluconeogenic enzymes are in the extracellular fraction/periplasm when cells are grown in media containing low glucose. Following a transfer of cells to high glucose media, their levels in the extracellular fraction are reduced rapidly. We hypothesized that changes in the secretome were not restricted to gluconeogenic enzymes. The goal of the current study was to use a proteomic approach to identify extracellular proteins whose levels changed when cells were transferred from low to high glucose media. We performed two iTRAQ experiments and identified 347 proteins that were present in the extracellular fraction including metabolic enzymes, proteins involved in oxidative stress, protein folding, and proteins with unknown functions. Most of these proteins did not contain typical ER-Golgi signal sequences. Moreover, levels of many of these proteins decreased upon a transfer of cells from media containing low to glucose media. Using an extraction procedure and Western blotting, we confirmed that the metabolic enzymes (glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, glucose-6-phosphate dehydrogenase, pyruvate decarboxylase), proteins involved in oxidative stress (superoxide dismutase and thioredoxin), and heat shock proteins (Ssa1p, Hsc82p, and Hsp104p) were in the extracellular fraction during growth in low glucose and that the levels of these extracellular proteins were reduced when cells were transferred to media containing high glucose. These proteins were associated with membranes in vesicle-enriched fraction. We also showed that small vesicles were present in the extracellular fraction in cells grown in low glucose. Following a transfer from low to high glucose media for 30 minutes, 98% of these vesicles disappeared from the extracellular fraction. Our data indicate that transferring cells from low to high glucose media induces a rapid decline in levels of a large number of extracellular proteins and the disappearance of small vesicles from the extracellular fraction. Therefore, we conclude that the secretome undergoes dynamic changes during transition from glucose-deficient to glucose-rich media. Most of these extracellular proteins do not contain typical ER signal sequences, suggesting that they are secreted via the non-classical pathway.
    Full-text · Article · Feb 2014 · Proteome Science
    0Comments 10Citations
    • "In S. cerevisiae, Ssp120 acted as a cargo-specific adaptor to interact with Emp46 and Emp47, which facilitate secretion by sorting cargo proteins into endoplasmic-reticulum-derived COPIIcoated vesicles, and participated in Golgi to endoplasmic reticulum recycling (Babu et al. 2012 ). Green fluorescent proteintagged Ssp120 in S. cerevisiae localized to early Golgi (Babu et al. 2012), while SsP120 of Kluyveromyces lactis could be detected in spent fermentation medium (Madinger et al. 2009), which implies that Ssp120 could be secreted out of cell wall into the medium. Ss-Caf1 contains a signal peptide, suggesting that it is likely a secreted protein. "
    [Show abstract] [Hide abstract] ABSTRACT: To decipher the mechanism of pathogenicity in Sclerotinia sclerotiorum, a pathogenicity-defective mutant, Sunf-MT6, was isolated from a T-DNA insertional library. Sunf-MT6 could not form compound appressorium and failed to induce lesions on leaves of rapeseed though it could produce more oxalic acid than the wild-type strain. However, it could enter into host tissues via wounds and cause typical necrotic lesions. Furthermore, Sunf-MT6 produced fewer but larger sclerotia than the wild-type strain Sunf-M. A gene, named Ss-caf1, was disrupted by T-DNA insertion in Sunf-MT6. Gene complementation and knockdown experiments confirmed that the disruption of Ss-caf1 was responsible for the phenotypic changes of Sunf-MT6. Ss-caf1 encodes a secretory protein with a putative Ca(2+)-binding EF-hand motif. High expression levels of Ss-caf1 were observed at an early stage of compound appressorium formation and in immature sclerotia. Expression of Ss-caf1 without signal peptides in Nicotiana benthamiana via Tobacco rattle virus-based vectors elicited cell death. These results suggest that Ss-caf1 plays an important role in compound appressorium formation and sclerotial development of S. sclerotiorum. In addition, Ss-Caf1 has the potential to interact with certain host proteins or unknown substances in host cells, resulting in subsequent host cell death.
    Full-text · Article · Jan 2014 · Molecular Plant-Microbe Interactions
    0Comments 7Citations
    • "But overall and with the exception of pathogens, the secretomes of yeasts obtained by physical or computational analyzes showed less heterogeneity than their fungal counterparts. Recently, the secretomes of several yeast species have been analyzed3031323334. A set of a dozen common proteins appears to be the fundamental unit of yeast secretomes consisting primarily of proteins involved in cell wall-related functions, including glucanases, transglucosylases , and chitinases. "
    [Show abstract] [Hide abstract] ABSTRACT: Microorganisms, although being very diverse because they comprise prokaryotic organisms such as bacteria or eukaryotic organisms such as fungi, all share an essential exodigester function. The consequence is their essential need to have a secretome adapted to their environment. The selection pressure exerted by environmental constraints led to the emergence of species with varying complexity in terms of composition of their secretomes. This review on fungal secretomes highlights the extraordinary variability among these organisms, even within the same species, and hence the absolute necessity to fully characterize all their components in the aims of understanding the fundamental mechanisms responsible for secretome plasticity and developing applications notably toward a better control of diseases caused by these pathogens.
    Full-text · Article · Feb 2013 · Proteomics
    0Comments 24Citations
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