A Role For Lte1p (a Low Temperature Essential Protein Involved in Mitosis) in Proprotein Processing in the Yeast Secretory Pathway

Abstract

We previously identified six single gene disruptions in Saccharomyces cerevisiae that allow enhanced immunoreactive insulin secretion primarily because of defective Kex2p-mediated endoproteolytic processing. Five eis mutants disrupted established VPS (vacuolar protein sorting) genes, The sixth, LTE1, is a Low Temperature (<15°C) Essential gene encoding a large protein with potential guanine nucleotide exchange (GEF) domains. Lte1p functions as a positive regulator of the mitotic GTPase Tem1p, and overexpression of Tem1p suppresses the low temperature mitotic defect of lte1. By sequence analysis, Tem1p has highest similarity to Vps21p (yeast homolog of mammalian Rab5). Unlike TEM1, LTE1 is not restricted to mitosis but is expressed throughout the cell cycle. Lte1p function in interphase cells is largely unknown. Here we confirm the eis phenotype of lte1 mutant cells and demonstrate a defect in proalpha factor processing that is rescued by expression of fulllength Lte1p but not a C-terminally truncated Lte1p lacking its GEF homology domain. Neither overexpression of Tem1p nor 13 other structurally related GTPases can suppress the secretory proprotein processing defect. However, overexpression of Vps21p selectively restores proprotein processing in a manner dependent upon the active GTP-bound form of the GTPase. By contrast, a vps21 mutant produces a synthetic defect with lte1 in proprotein processing, as well as a synthetic growth defect. Together, the data underscore a link between the mitotic regulator, Lte1p, and protein processing and trafficking in the secretory/endosomal system.

Molecular quantification of Saccharomyces cerevisiae α-pheromone secretion

“…Assays based on growth inhibition are highly sensitive to variation in temperature, incubation time, medium depth and composition, and the number of MATa cells plated. Furthermore, most halo assays measure the amount of pheromone secreted over the course of 1 or 2 days by a concentrated patch of MATa cells (Kurjan, 1985;Zhao et al, 2007;Banuelos et al, 2010;Gharakhanian et al, 2011;Ricarte et al, 2011;Corbacho et al, 2012); any difference in growth rate between strains will have a large effect on halo size (Smith & Greig, 2010). Using the activities of recombinant proteins as proxy measures for a-pheromone secretion allows more precise quantification, but may not accurately reflect the normal behaviour of the nonmanipulated system.…”

“…This response provides the basis for a widespread qualitative measure of pheromone production called a 'halo' assay; pheromone spotted onto a lawn of cells of the opposite mating type produces a zone of growth inhibition proportional to the amount of pheromone spotted. While halo assays are simple and effective for identifying mutations resulting in extreme secretion defects (Zhao et al, 2007;Banuelos et al, 2010;Gharakhanian et al, 2011;Ricarte et al, 2011;Corbacho et al, 2012), a more sensitive and quantifiable technique is required to improve not only our understanding of the fine-scale genetic and physiological determinants of secretion (c.f. Idris et al, 2010), but also the evolutionary forces shaping intercellular signalling (Krakauer & Pagel, 1996).…”

Analysis of low temperature-induced genes (LTIG) in wine yeast during alcoholic fermentation

“…A total of 10 cold-shock or low temperature-induced genes (LTIG) were analyzed. These included HSP104, HSP26, and TCP1, which encode molecular chaperones that are involved in protein folding or protein aggregation prevention (Ursic & Culbertson, 1991;Haslbeck et al, 1999;Bösl et al, 2006); HSP12, TIP1, and TIR2, which encode proteins involved in the maintenance of the plasma membrane and cell wall (Kondo & Inouye, 1991;Sales et al, 2000;Abramova et al, 2001); NSR1 and LOT2 (RPL2B), which are involved in pre-rRNA process-ing and ribosome biogenesis (Kondo & Inouye, 1992;Zhang et al, 2001); LTE1, which encodes the Cdc25 family guanine-nucleotide-exchanging factors (Zhao et al, 2007); and CSF1, which was described by Tokai et al (2000) to associate with the nutrient transport system and is only required for fermentation at low temperature.…”

Recent advances in bio‐chemical, molecular and physiological aspects of membrane lipid derivatives in plant pathology

“…Genetic studies have indicated that PLD and phosphatidic acid participate in regulating the physiological processes in plant-pathogen interactions and other biotic stresses (Bargmann & Munnik, 2006;Wang et al, 2006). (Zhao, Chang, Toh, & Arvan, 2007). Phosphatidic acid regulates phosphorylation of proteins, translation and transcription in biosynthesis of glycolipids, proliferation of cells and growth (Huang & Frohman, 2007;Nadeem et al, 2019;Nadeem et al, 2020).…”

“…They primarily circulate the information from G‐protein‐coupled receptors on the cellular membrane to the internal structure of cells for biochemical regulation of cell defense functions. They also function in the modulation of cell physiological and phenotypic outcomes such as disease susceptibility or resistance (Zhao, Chang, Toh, & Arvan, 2007). Phosphatidic acid regulates phosphorylation of proteins, translation and transcription in biosynthesis of glycolipids, proliferation of cells and growth (Huang & Frohman, 2007; Nadeem et al, 2019; Nadeem et al, 2020).…”