Sodium-dependent neutral amino acid transporter B(0)AT1 (SLC6A19) and imino acid (proline) transporter SIT1 (SLC6A20) are expressed at the luminal membrane of small intestine enterocytes and proximal tubule kidney cells where they exert key functions for amino acid (re)absorption as documented by their role in Hartnup disorder and iminoglycinuria, respectively. Expression of B(0)AT1 was shown in rodent intestine to depend on the presence of the carboxypeptidase angiotensin-converting enzyme 2 (ACE2). This enzyme belongs to the renin-angiotensin system and its expression is induced by treatment with ACE-inhibitors (ACEIs) or angiotensin II AT1 receptor blockers (ARBs) in many rodent tissues. We show here in the Xenopus laevis oocyte expression system that human ACE2 also functionally interacts with SIT1. To investigate in human intestine the potential effect of ACEIs or ARBs on ACE2, we analysed intestinal biopsies taken during routine gastroduodenoscopy and ileocolonoscopy from 46 patients of which 9 were under ACEI and 13 ARB treatment. Analysis of transcript expression by real-time PCR and of proteins by immunofluorescence showed a co-localization of SIT1 and B(0)AT1 with ACE2 in the brush-border membrane of human small intestine enterocytes and a distinct axial expression pattern of the tested gene products along the intestine. Patients treated with ACEIs displayed in comparison with untreated controls increased intestinal mRNA levels of ACE2, peptide transporter PEPT1 (SLC15A1) and AA transporters B(0)AT1 and PAT1 (SLC36A1). This study unravels in human intestine the localization and distribution of intestinal transporters involved in amino acid absorption and suggests that ACEIs impact on their expression. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64

Short-lived proteins are degraded by proteasome complexes, which contain a proteolytic core particle (CP) but differ in the number of regulatory particles (RPs) and activators. A recently described member of conserved proteasome activators is Blm10. Blm10 contains 32 HEATlike modules and is structurally related to the nuclear import receptor importin/karyopherin b. In proliferating yeast, RP-CP assemblies are primarily nuclear and promote cell division. During quiescence, RP-CP assemblies dissociate and CP and RP are sequestered into motile cytosolic proteasome storage granuli (PSG). Here, we show that CP sequestration into PSG depends on Blm10, whereas RP sequestration into PSG is independent of Blm10. PSG rapidly clear upon the resumption of cell proliferation and proteasomes are relocated into the nucleus. Thereby, Blm10 facilitates nuclear import of CP. Blm10-bound CP serves as an import receptor-cargo complex, as Blm10 mediates the interaction with FG-rich nucleoporins and is dissociated from the CP by Ran-GTP. Thus, Blm10 represents the first CP-dedicated nuclear import receptor in yeast.

Proteasomes are key protease complexes responsible for protein degradation, and their localization changes with the growth conditions. This work in yeast shows that proteasomes exit the nucleus with the transition from proliferation to quiescence. Ubiquitin is a key player in proteasome dynamics and cytoplasmic proteasome granule formation.

Absorption of neutral amino acids across the luminal membrane of intestinal enterocytes is mediated by the broad neutral amino acid transporter B 0 AT1 (SLC6A19). Its intestinal expression depends on co-expression of the membrane-anchored peptidase angiotensin converting enzyme 2 (ACE2) and is additionally enhanced by aminopeptidase N (CD13). We investigated in this study the expression of B 0 AT1 and its auxiliary peptidases as well as its transport function along the rat small intestine. Additionally, we tested its possible shortand long-term regulation by dietary proteins and amino acids. We showed by immunofluorescence that B 0 AT1, ACE2 and CD13 co-localize on the luminal membrane of small intestinal villi and by Western blotting that their protein expression increases in distal direction. Furthermore, we observed an elevated transport activity of the neutral amino acid L-isoleucine during the nocturnal active phase compared to the inactive one. protein expression under amino acid-supplemented diet in the proximal section but not in the distal one and for ACE2 protein expression a reverse localization of the effect. Dietary regulation for CD13 protein expression was not as distinct as for the two other proteins. Ring uptake experiments showed a tendency for increased L-isoleucine uptake under amino acid-supplemented diet and in vivo L-isoleucine absorption was more efficient under high protein and amino acid-supplemented diet. Additionally, plasma levels of branched-chain amino acids were elevated under high protein and amino acid diet. Taken together, our experiments did not reveal an acute amino acid-induced regulation of B 0 AT1 but revealed a chronic dietary adaptation mainly restricted to the proximal segment of the small intestine.

Basolateral efflux is a crucial step for amino acid (AA) (re)absorption across small intestine and kidney proximal tubule epithelia mediated by various transporters. There are uniporters that mediate the facilitated diffusion of essential AAs, as does for instance aromatic AA transporter TAT1 (Slc16a10) and also antiporters such as LAT2‐4F2hc (SLC7A8‐SLC3A2) that exchanges neutral AAs. To test the hypothesis that the recycling of aromatic AAs via TAT1 allows the vectorial efflux of other AAs via obligatory exchanger LAT2‐4F2hc, LAT2‐/‐ TAT1‐/‐ double knockout (dKO) mice were generated. These mice have a reduced body weight (~‐17% at 3 months) but no other overt phenotypic alteration. Under normal protein diet, they excrete in the urine, compared to LAT2‐/‐ and TAT1‐/‐ knockout mice, higher amounts of aromatic AAs and of some other AAs that are not substrates of TAT1. The amino aciduria was further increased under high protein diet and involved all proteogenic AAs but some charged ones. Screening the mRNA levels of ~20 AA transporters in the kidney suggested a trend of compensatory changes as the level of the transcript of 5 basolateral AA transporters was increased in dKO mice under high protein diet. Preliminary transport experiments with small intestine gut sacs confirm the alteration of transepithelial transport. These observations support the hypothesis that the basolateral efflux of LAT2 substrates depends on the recycling of aromatic AAs via TAT1.Supported by Swiss National Science Foundation.

To ensure body homeostasis amino acids (AA) are (re)absorbed by epithelial cells of the intestinal mucosa and renal proximal tubules. This sequential transepithelial transport includes an apical uptake followed by a basolateral efflux. The luminal uptake is mediated by different AA transporters but most neutral AAs are transported by the luminal Broad neutral Amino acid Transporter B0AT1 (SLC6A19).The regulation of this transporter is poorly known; in particular its potential short‐term regulation by its substrates has not been investigated. We hypothesized that similarly to glucose transporters the expression of B0AT1 might be repressed if costs of its synthesis and expression exceed its benefits and thus it would be upregulated by AAs to absorb them efficiently when available. To investigate the possible short‐term regulation of B0AT1 by AAs in vivo, we assessed in rats whether after a fast of 4 or 16 hours, intragastric AA application upregulates B0AT1 expression in intestinal brush border membranes. The time point of analysis after amino acid gavage was determined based on micro Computer Tomography measurements showing when the AA mix reached the small intestine. However, functional analysis of AA absorption in isolated ex vivo intestinal rings showed no significant difference between rats having received a water or an AA gavage. In addition, protein expression levels of B0AT1 in the intestinal brush border analyzed by Western blotting did not reveal a difference between rats having received a water or AA gavage. In conclusion, in the selected experimental conditions, the expression and function of the apical AA transporter B0AT1 was not regulated in the short‐term by the preceding application of its substrate AAs.Supported by the Swiss National Science Foundation.