Background
The Saccharomyces cerevisiae Jen1 transporter mediates electroneutral proton symport of lactate and pyruvate. In lactate-grown batch cultures, growth-coupled medium alkalinization was previously shown to coincide with endocytosis of Jen1.

Results
To investigate the physiological relevance of pH-dependent Jen1 endocytosis, S. cerevisiae was grown in carbon-limited continuous cultures on a mixed ethanol-lactate feed. When applying a linearly increasing pH (6.75–7.25) to these cultures, lactate and pyruvate concentrations in the external medium progressively increased. Up to a culture pH of 7.0, these extracellular concentrations aligned with a predicted thermodynamic equilibrium of reversible, Jen1-mediated electroneutral carboxylate-proton symport. Consistent with earlier reports, pronounced Jen1 internalization occurred above pH 7.0. At these mildly alkaline pH values, a more pronounced increase of residual lactate concentrations and transcriptional upregulation of genes involved in oxidative phosphorylation were consistent with increased cellular energy demands.

Conclusion
This study reveals how pH-dependent regulation of carboxylate transporters shapes cellular adaptation to changing environmental conditions. Insights into these regulatory mechanisms can inform strategies to optimize microbial cell factories operating under variable pH regimes in industrial settings. The integrated analysis of transport, Jen1 localization, and transcriptional responses in growing continuous cultures uncovered physiological challenges associated with electroneutral carboxylate/proton symport under mildly alkaline conditions. The data support the hypothesis that Jen1 internalization evolved to prevent intracellular metabolite loss under unfavorable pH conditions.

Plasma membrane transporters play pivotal roles in the import of nutrients, including sugars, amino acids, nucleobases, carboxylic acids, and metal ions, that surround fungal cells. The selective removal of these transporters by endocytosis is one of the most important regulatory mechanisms that ensures a rapid adaptation of cells to the changing environment (e.g., nutrient fluctuations or different stresses). At the heart of this mechanism lies a network of proteins that includes the arrestin‐related trafficking adaptors (ARTs) which link the ubiquitin ligase Rsp5 to nutrient transporters and endocytic factors. Transporter conformational changes, as well as dynamic interactions between its cytosolic termini/loops and with lipids of the plasma membrane, are also critical during the endocytic process. Here, we review the current knowledge and recent findings on the molecular mechanisms involved in nutrient transporter endocytosis, both in the budding yeast Saccharomyces cerevisiae and in some species of the filamentous fungus Aspergillus. We elaborate on the physiological importance of tightly regulated endocytosis for cellular fitness under dynamic conditions found in nature and highlight how further understanding and engineering of this process is essential to maximize titer, rate and yield (TRY)-values of engineered cell factories in industrial biotechnological processes.

In Saccharomyces cerevisiae, the complete set of proteins involved in transport of lactic acid across the cell membrane has not been determined. In this study, we aimed to identify transport proteins not previously described to be involved in lactic acid transport via a combination of directed evolution, whole-genome resequencing and reverse engineering. Evolution of a strain lacking all known lactic acid transporters on lactate led to the discovery of mutated Ato2 and Ato3 as two novel lactic acid transport proteins. When compared to previously identified S. cerevisiae genes involved in lactic acid transport, expression of ATO3T284C was able to facilitate the highest growth rate (0.15 ± 0.01 h-1) on this carbon source. A comparison between (evolved) sequences and 3D models of the transport proteins showed that most of the identified mutations resulted in a widening of the narrowest hydrophobic constriction of the anion channel. We hypothesize that this observation, sometimes in combination with an increased binding affinity of lactic acid to the sites adjacent to this constriction, are responsible for the improved lactic acid transport in the evolved proteins.