‘Transient’ genetic suppression facilitates generation of hexose transporter null mutants inLeishmania mexicana

The Hxt2 glucose transport protein of Saccharomyces cerevisiae was genetically fused at its C-terminus with the green fluorescent protein (GFP). The Hxt2-GFP fusion protein is a functional hexose transporter: it restored growth on glucose to a strain bearing null mutations in the hexose transporter genes GAL2 and HXT1 to HXT7. Furthermore, its glucose transport activity in this null strain was not markedly different from that of the wild-type Hxt2 protein. We calculated from the fluorescence level and transport kinetics that induced cells had 1.4×105 Hxt2-GFP molecules per cell, and that the catalytic-centre activity of the Hxt2-GFP molecule in vivo is 53 s-1 at 30 °C. Expression of Hxt2-GFP was induced by growth at low concentrations of glucose. Under inducing conditions the Hxt2-GFP fluorescence was localized to the plasma membrane. In a strain impaired in the fusion of secretory vesicles with the plasma membrane, the fluorescence accumulated in the cytoplasm. When induced cells were treated with high concentrations of glucose, the fluorescence was redistributed to the vacuole within 4 h. When endocytosis was genetically blocked, the fluorescence remained in the plasma membrane after treatment with high concentrations of glucose.

Download Full-text

Caenorhabditis elegans Junctophilin has tissue-specific functions and regulates neurotransmission with extended-synaptotagmin

Genetics ◽

10.1093/genetics/iyab063 ◽

2021 ◽

Author(s):

Christopher A Piggott ◽

Zilu Wu ◽

Stephen Nurrish ◽

Suhong Xu ◽

Joshua M Kaplan ◽

...

Keyword(s):

Calcium Channels ◽

Calcium Channel ◽

Tissue Specific ◽

Voltage Gated ◽

Membrane Contact Sites ◽

Contact Sites ◽

Pharyngeal Muscles ◽

Membrane Contact ◽

Null Mutants

Abstract The junctophilin family of proteins tether together plasma membrane (PM) and endoplasmic reticulum (ER) membranes, and couple PM- and ER-localized calcium channels. Understanding in vivo functions of junctophilins is of great interest for dissecting the physiological roles of ER-PM contact sites. Here, we show that the sole C. elegans junctophilin JPH-1 localizes to discrete membrane contact sites in neurons and muscles and has important tissue-specific functions. jph-1 null mutants display slow growth and development due to weaker contraction of pharyngeal muscles, leading to reduced feeding. In the body wall muscle, JPH-1 co-localizes with the PM-localized EGL-19 voltage-gated calcium channel and ER-localized UNC-68/RyR calcium channel, and is required for animal movement. In neurons, JPH-1 co-localizes with the membrane contact site protein Extended-SYnaptoTagmin 2 (ESYT-2) in soma, and is present near presynaptic release sites. Interestingly, jph-1 and esyt-2 null mutants display mutual suppression in their response to aldicarb, suggesting that JPH-1 and ESYT-2 have antagonistic roles in neuromuscular synaptic transmission. Additionally, we find an unexpected cell non-autonomous effect of jph-1 in axon regrowth after injury. Genetic double mutant analysis suggests that jph-1 functions in overlapping pathways with two PM-localized voltage-gated calcium channels, egl-19 and unc-2, and unc-68/RyR for animal health and development. Finally, we show that jph-1 regulates the colocalization of EGL-19 and UNC-68 and that unc-68/RyR is required for JPH-1 localization to ER-PM puncta. Our data demonstrate important roles for junctophilin in cellular physiology, and also provide insights into how junctophilin functions together with other calcium channels in vivo.

Download Full-text

Molecular Basis of Fructose Utilization by the Wine Yeast Saccharomyces cerevisiae: a Mutated HXT3 Allele Enhances Fructose Fermentation

Applied and Environmental Microbiology ◽

10.1128/aem.02269-06 ◽

2007 ◽

Vol 73 (8) ◽

pp. 2432-2439 ◽

Cited By ~ 67

Author(s):

Carole Guillaume ◽

Pierre Delobel ◽

Jean-Marie Sablayrolles ◽

Bruno Blondin

Keyword(s):

Saccharomyces Cerevisiae ◽

Molecular Basis ◽

Alcoholic Fermentation ◽

Wine Yeast ◽

Glycolytic Flux ◽

Hexose Transporter ◽

Wine Yeasts ◽

Yeast Saccharomyces Cerevisiae ◽

High Fructose ◽

Fructose Fermentation

ABSTRACT Fructose utilization by wine yeasts is critically important for the maintenance of a high fermentation rate at the end of alcoholic fermentation. A Saccharomyces cerevisiae wine yeast able to ferment grape must sugars to dryness was found to have a high fructose utilization capacity. We investigated the molecular basis of this enhanced fructose utilization capacity by studying the properties of several hexose transporter (HXT) genes. We found that this wine yeast harbored a mutated HXT3 allele. A functional analysis of this mutated allele was performed by examining expression in an hxt1-7Δ strain. Expression of the mutated allele alone was found to be sufficient for producing an increase in fructose utilization during fermentation similar to that observed in the commercial wine yeast. This work provides the first demonstration that the pattern of fructose utilization during wine fermentation can be altered by expression of a mutated hexose transporter in a wine yeast. We also found that the glycolytic flux could be increased by overexpression of the mutant transporter gene, with no effect on fructose utilization. Our data demonstrate that the Hxt3 hexose transporter plays a key role in determining the glucose/fructose utilization ratio during fermentation.

Download Full-text