While several clathrin-independent endocytic processes have been described so far, their biological relevance often remains elusive, especially in pathophysiological contexts such as cancer. In this ...study, we find that the tumor marker CD166/ALCAM (Activated Leukocyte Cell Adhesion Molecule) is a clathrin-independent cargo. We show that endophilin-A3-but neither A1 nor A2 isoforms-functionally associates with CD166-containing early endocytic carriers and physically interacts with the cargo. Our data further demonstrates that the three endophilin-A isoforms control the uptake of distinct subsets of cargoes. In addition, we provide strong evidence that the construction of endocytic sites from which CD166 is taken up in an endophilin-A3-dependent manner is driven by extracellular galectin-8. Taken together, our data reveal the existence of a previously uncharacterized clathrin-independent endocytic modality, that modulates the abundance of CD166 at the cell surface, and regulates adhesive and migratory properties of cancer cells.
Cases of congenital disorders of glycosylation (CDG) have been associated with specific mutations within the gene encoding the human Golgi TMEM165 (transmembrane protein 165), belonging to UPF0016 ...(uncharacterized protein family 0016), a family of secondary ion transporters. To date, members of this family have been reported to be involved in calcium, manganese, and pH homeostases. Although it has been suggested that TMEM165 has cation transport activity, direct evidence for its Ca2+- and Mn2+-transporting activities is still lacking. Here, we functionally characterized human TMEM165 by heterologously expressing it in budding yeast (Saccharomyces cerevisiae) and in the bacterium Lactococcus lactis. Protein production in these two microbial hosts was enhanced by codon optimization and truncation of the putatively autoregulatory N terminus of TMEM165. We show that TMEM165 expression in a yeast strain devoid of Golgi Ca2+ and Mn2+ transporters abrogates Ca2+- and Mn2+-induced growth defects, excessive Mn2+ accumulation in the cell, and glycosylation defects. Using bacterial cells loaded with the fluorescent Fura-2 probe, we further obtained direct biochemical evidence that TMEM165 mediates Ca2+ and Mn2+ influxes. We also used the yeast and bacterial systems to evaluate the impact of four disease-causing missense mutations identified in individuals with TMEM165-associated CDG. We found that a mutation leading to a E108G substitution within the conserved UPF0016 family motif significantly reduces TMEM165 activity. These results indicate that TMEM165 can transport Ca2+ and Mn2+, which are both required for proper protein glycosylation in cells. Our work also provides tools to better understand the pathogenicity of CDG-associated TMEM165 mutations.
The gradual acidification of the secretory pathway is conserved and extremely important for eukaryotic cells, but until now there was no pH sensor available to monitor the pH of the early Golgi ...apparatus in Saccharomyces cerevisiae. Therefore, we developed a pHluorin-based sensor for in vivo measurements in the lumen of the Golgi. By using this new tool we show that the cis- and medial-Golgi pH is equal to 6.6-6.7 in wild type cells during exponential phase. As expected, V-ATPase inactivation results in a near neutral Golgi pH. We also uncover that surprisingly Vph1p isoform of the V-ATPase is prevalent to Stv1p for Golgi acidification. Additionally, we observe that during changes of the cytosolic pH, the Golgi pH is kept relatively stable, mainly thanks to the V-ATPase. Eventually, this new probe will allow to better understand the mechanisms involved in the acidification and the pH control within the secretory pathway.
Competence for DNA transformation is a major strategy for bacterial adaptation and survival. Yet, this successful tactic is energy-consuming, shifts dramatically the metabolism, and transitory ...impairs the regular cell-cycle. In streptococci, complex regulatory pathways control competence deactivation to narrow its development to a sharp window of time, a process known as competence shut-off. Although characterized in streptococci whose competence is activated by the ComCDE signaling pathway, it remains unclear for those controlled by the ComRS system. In this work, we investigate competence shut-off in the major human gut commensal Streptococcus salivarius. Using a deterministic mathematical model of the ComRS system, we predicted a negative player under the control of the central regulator ComX as involved in ComS/XIP pheromone degradation through a negative feedback loop. The individual inactivation of peptidase genes belonging to the ComX regulon allowed the identification of PepF as an essential oligoendopeptidase in S. salivarius. By combining conditional mutants, transcriptional analyses, and biochemical characterization of pheromone degradation, we validated the reciprocal role of PepF and XIP in ComRS shut-off. Notably, engineering cleavage site residues generated ultra-resistant peptides producing high and long-lasting competence activation. Altogether, this study reveals a proteolytic shut-off mechanism of competence in the salivarius group and suggests that this mechanism could be shared by other ComRS-containing streptococci.
Yeast cells, to be able to grow on a wide variety of nitrogen sources, regulate the set of nitrogen transporters present at their plasma membrane. Such regulation relies on both transcriptional and ...post-translational events. Although microarray studies have identified most nitrogen-sensitive genes, nitrogen-induced post-translational regulation has only been studied for very few proteins among which the general amino acid permease Gap1. Adding a preferred nitrogen source to proline-grown cells triggers Gap1 endocytosis and vacuolar degradation in an Rsp5-Bul1/2-dependent manner. Here, we used a proteomic approach to follow the dynamics of the plasma membrane proteome after addition of a preferred nitrogen source. We identified new targets of the nitrogen regulation and four transporters of poor nitrogen sources—Put4, Opt2, Dal5, and Ptr2—that rapidly decrease in abundance. Although the kinetics is different for each transporter, we found that three of them—Put4, Dal5, and Ptr2—are endocytosed, like Gap1, in an Rsp5-dependent manner and degraded in the vacuole. Finally, we showed that Gap1 stabilization at the plasma membrane, through deletion of Bul proteins, regulates the abundance of Put4, Dal5 and Ptr2.
Congenital disorders of glycosylation (CDG) are severe inherited diseases in which aberrant protein glycosylation is a hallmark. From this genetically and clinically heterogenous group, a significant ...subgroup due to Golgi homeostasis defects is emerging. We previously identified TMEM165 as a Golgi protein involved in CDG. Extremely conserved in the eukaryotic reign, the molecular mechanism by which TMEM165 deficiencies lead to Golgi glycosylation abnormalities is enigmatic. AsGDT1 is the ortholog of TMEM165 in yeast, both gdt1Δ null mutant yeasts and TMEM165 depleted cells were used. We highlighted that the observed Golgi glycosylation defects due to Gdt1p/TMEM165 deficiency result from Golgi manganese homeostasis defect. We discovered that in both yeasts and mammalian Gdt1p/TMEM165-deficient cells, Mn(2+) supplementation could restore a normal glycosylation. We also showed that the GPP130 Mn(2+) sensitivity was altered in TMEM165 depleted cells. This study not only provides novel insights into the molecular causes of glycosylation defects observed in TMEM165-deficient cells but also suggest that TMEM165 is a key determinant for the regulation of Golgi Mn(2+) homeostasis.
• The photosynthetic machinery of plants must be regulated to maximize the efficiency of light reactions and CO₂ fixation. Changes in free Ca2+ in the stroma of chloroplasts have been observed at the ...transition between light and darkness, and also in response to stress stimuli. Such Ca2+ dynamics have been proposed to regulate photosynthetic capacity. However, the molecular mechanisms of Ca2+ fluxes in the chloroplasts have been unknown.
• By employing a Ca2+ reporter-based approach, we identified two chloroplast-localized Ca2+ transporters in Arabidopsis thaliana, BICAT1 and BICAT2, that determine the amplitude of the darkness-induced Ca2+ signal in the chloroplast stroma.
• BICAT2 mediated Ca2+ uptake across the chloroplast envelope, and its knockout mutation strongly dampened the dark-induced Ca2+stroma signal. Conversely, this Ca2+ transient was increased in knockout mutants of BICAT1, which transports Ca2+ into the thylakoid lumen. Knockout mutation of BICAT2 caused severe defects in chloroplast morphology, pigmentation and photosynthetic light reactions, rendering bicat2 mutants barely viable under autotrophic growth conditions, while bicat1 mutants were less affected.
• These results show that BICAT transporters play a role in chloroplast Ca2+ homeostasis. They are also involved in the regulation of photosynthesis and plant productivity. Further work will be required to reveal whether the effect on photosynthesis is a direct result of their role as Ca2+ transporters.
The UPF0016 family is a group of uncharacterized membrane proteins, well conserved through evolution and defined by the presence of one or two copies of an E-Φ-G-D-(KR)-(ST) consensus motif. Our ...previous results have shown that two members of this family, the human TMEM165 and the budding yeast Gdt1p, are functionally related and might form a new group of cation/Ca2+ exchangers. Most members of the family are made of two homologous clusters of three transmembrane spans, separated by a central loop and assembled with an opposite orientation in the membrane. However, some bacterial members of the family have only one cluster of transmembrane domains. Among these 'single-domain membrane proteins' some cyanobacterial members were found as pairs of adjacent genes within the genome, but each gene was slightly different. We performed a bioinformatic analysis to propose the molecular evolution of the UPF0016 family and the emergence of the antiparallel topology. Our hypotheses were confirmed experimentally using functional complementation in yeast. This suggests an important and conserved function for UPF0016 proteins in a fundamental cellular process. We also show that members of the UPF0016 family share striking similarities, but no primary sequence homology, with members of the cation/Ca2+ exchangers (CaCA) superfamily. Such similarities could be an example of convergent evolution, supporting the previous hypothesis that members of the UPF0016 family are cation/Ca2+ exchangers.
Defects in the human protein TMEM165 are known to cause a subtype of Congenital Disorders of Glycosylation. Transmembrane protein 165 (TMEM165) belongs to an uncharacterized family of membrane ...proteins called Uncharacterized Protein Family 0016, which are well conserved throughout evolution and share characteristics reminiscent of the cation/Ca²⁺ exchanger superfamily. Gcr1 dependent translation factor 1 (Gdt1p), the budding yeast member of this family, contributes to Ca²⁺ homeostasis via an uncharacterized Ca²⁺ transport pathway localized in the Golgi apparatus. The gdt1∆ mutant was found to be sensitive to high concentrations of Ca²⁺, and interestingly, this sensitivity was suppressed by expression of TMEM165, the human ortholog of Gdt1p, indicating conservation of function among the members of this family. Patch-clamp analyses on human cells indicated that TMEM165 expression is linked to Ca²⁺ ion transport. Furthermore, defects in TMEM165 affected both Ca²⁺ and pH homeostasis. Based on these results, we propose that Gdt1p and TMEM165 could be members of a unique family of Golgi-localized Ca²⁺/H⁺ antiporters and that modification of the Golgi Ca²⁺ and pH balance could explain the glycosylation defects observed in TMEM165-deficient patients.
Endocytosis of membrane proteins in yeast requires α-arrestin-mediated ubiquitylation by the ubiquitin ligase Rsp5. Yet, the diversity of α-arrestin targets studied is restricted to a small subset of ...plasma membrane (PM) proteins. Here, we performed quantitative proteomics to identify new targets of 12 α-arrestins and gained insight into the diversity of pathways affected by α-arrestins, including the cell wall integrity pathway and PM-endoplasmic reticulum contact sites. We found that Art2 is the main regulator of substrate- and stress-induced ubiquitylation and endocytosis of the thiamine (vitamin B1) transporters: Thi7, nicotinamide riboside transporter 1 (Nrt1), and Thi72. Genetic screening allowed for the isolation of transport-defective Thi7 mutants, which impaired thiamine-induced endocytosis. Coexpression of inactive mutants with wild-type Thi7 revealed that both transporter conformation and transport activity are important to induce endocytosis. Finally, we provide evidence that Art2 mediated Thi7 endocytosis is regulated by the target of rapamycin complex 1 (TORC1) and requires the Sit4 phosphatase but is not inhibited by the Npr1 kinase.
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