Increased endocytosis in the Saccharomyces cerevisiae fragile mutant VY1160

Abstract

The VY1160 mutant is characterized by cell lysis in hypotonic solutions and generally increased permeability to substances for which Saccharomyces cerevisiae cells are not permeable. Two mutations, srb1 and ts1, have been identified in VY1160 mutant, and previous studies (Kozhina et al., 1979) have shown srb1 to be responsible for cell lysis. We now present evidence that the ts1 mutation leads to increased endocytosis in VY1160 cells. The internalization of lucifer yellow carbohydrazide in VY1160 cells is time-, temperature- and energy-dependent and consistent with a fluid-phase mechanism of endocytosis. The rate of steady-state accumulation of the dye at 37 degrees C is 145 ng/micrograms DNA per h for VY1160 mutant and 23 ng/micrograms DNA per h for S288C parental strain. Studies with isogenic strains having either the srb1 or the ts1 mutation, or SRB1 TS1 wild-type alleles have shown that only ts1 strains possess increased endocytosis. Quantitation of endocytosis in cells grown at 24 degrees C and shifted at 38 degrees C shows that ts1 strains, but not srb1 and wild-type strains, increase ten-fold the internalization of lucifer yellow 2 h after the shift at 38 degrees C. The analysis of ts1 x wild-type crosses provides evidence that the temperature-sensitive phenotype segregates together with the enhanced endocytosis. It is concluded that the increased endocytosis might explain the generally increased permeability of VY1160 mutant cells.

The low-affinity component of Saccharomyces cerevisiae maltose transport is an artifact

“…This low concentration is not consistent with any of the uptake mechanisms reported in yeast cells (4) and suggests that the activity ascribed to the low-affinity component of the maltose transport system is due not to a transport process but to trapping in the periplasmic space. A trapping with similar characteristics has been reported for lucifer yellow, a hydrophilic solute that does not diffuse spontaneously through the plasma membrane (19,22). In this case, it has been shown by fluorescence microscopy that yeast cells retain the solute in the periplasmic space, most likely through nonspecific binding to the cell wall and plasma membrane, until a plateau is reached in ca.…”

A Neurotoxic Phospholipase A2 Impairs Yeast Amphiphysin Activity and Reduces Endocytosis

“…The uptake rate of the fluid-phase endocytosis marker Lucifer Yellow (Lucifer Yellow CH dilithium salt, Sigma) in wild-type, rvs161Δ , rvs167Δ and rvs161Δ rvs167Δ strains expressing AtxA and corresponding control strains was quantified as described [24], [85]. Briefly, cells were grown to mid-logarithmic phase, harvested and resuspended in fresh medium containing 4 mg/ml LY and 20 mM NaN 3 to prevent cell growth during LY staining.…”

Involvement of endocytosis in catabolite inactivation of the K+ and glucose transport systems in Saccharomyces cerevisiae

“…Two temperature-sensitive strains carrying the tsl mutation that leads to increased rate of fluidphase endocytosis [7] have been used in this work as well as a TS1 strain obtained by transformation of a tsl mutant with an integrative plasmid carrying the TS1 gene [8]. The tsl mutants showed, at 35°C, an increased internalization of LY as compared with the wild-type or the transformed cells whereas at 24°C internalization of the dye was similar in all tested strains (results not shown).…”

“…(MATa ura3 TS1) [7], 54P (MATa ura3 tsl) [7], SSDI 03 78-1097(94)00243 -K 78 H17 (MATa ura3 tsl leu2) (P. Venkov, personal communication); and TY17, obtained by transformation of H17 with the plasmid YIp-TS1 (MATa URA3 TS1 leu2) [8]. Cells were grown aerobically at 24°C in YPD (1% yeast extract, 2% peptone, and 2% glucose, w/v).…”

“…To express the tsl mutation, the cells were harvested by centrifugation during exponential growth, suspended in three times the initial volume of flesh medium and incubated at 35°C for 2 h [7]. Controls at 24°C were run in parallel.…”