Interactions of cytosolic tails in the Jen1 carboxylate transporter are critical for trafficking and transport activity

Abstract

Plasma membrane (PM) transporters of the major facilitator superfamily (MFS) are essential for cell metabolism, growth and response to stress or drugs. In Saccharomyces cerevisiae, Jen1 is a monocarboxylate/H+ symporter that provides a model to dissect the molecular details underlying cellular expression, transport mechanism and turnover of MFS transporters. Here, we present evidence revealing novel roles of the cytosolic N- and C- termini of Jen1 in its biogenesis, PM stability and transport activity, using functional analyses of Jen1 truncations and chimeric constructs with UapA, an endocytosis-insensitive transporter of Aspergillus nidulans. Our results show that both N- and C-termini are critical for Jen1 trafficking to the PM, transport activity and endocytosis. Importantly, we provide evidence that Jen1 N- and C- termini undergo transport-dependent dynamic intra-molecular interactions, which affect the transport activity and turnover of Jen1. Our results support an emerging concept where the cytoplasmic termini of PM transporters control transporter cell-surface stability and function through flexible intra-molecular interactions with each other. These findings may be extended to other MFS members to understand conserved and evolving mechanisms underlying transporter structure-function relationships.

Optimization of the fluorogen-activating protein tag for quantitative protein trafficking and co-localization studies in S. cerevisiae

“…Considering that three α-arrestins mediate the internalization of Ste2 and Ste3 ( i.e., Rod1, Rog3 and Art1 or Aly1, Aly2 and Art1, respectively), there is opportunity for both a common regulatory sequence ( i.e., for Art1) and divergent regulatory sequences ( i.e., for Rod1/Rog3 vs Aly1/Aly2) in Ste2 and Ste3. To date, only a few α-arrestin interaction interfaces have been defined for any membrane protein, but based on these that are defined it appears that α-arrestins preferentially associate with acidic patches on cargo proteins (Lin et al ., 2008; Wawrzycka et al ., 2019; Ivashov et al ., 2020; Barata-Antunes et al ., 2022). Our earlier work demonstrated that α-arrestins can bind the C-tails of Ste3 and Ste2, suggesting that key interactions needed to recruit α-arrestins occur in these regions (Alvaro et al ., 2014; Prosser et al ., 2015).…”

Endocytosis of the plasma membrane carboxylate transporter Jen1 inSaccharomyces cerevisiaeunder alkali stress: a mechanism for preventing loss of essential metabolites

“…Jen1 was also reported to transport selenite [12] and it is expressed and localized at the PM when cells are growing on non-fermentable carbon sources, including lactate, pyruvate, acetate and ethanol [13]. A variety of signals induce Jen1 internalization and degradation, including glucose [10,[14][15][16][17][18][19][20], rapamycin, and cycloheximide ( [21], see also [5] for a review). In our recent study [21],…”