Expression and regulation of the neutral amino acid transporter B0AT1 in rat small intestine.

Absorption of neutral amino acids across the luminal membrane of intestinal enterocytes is mediated by the broad neutral amino acid transporter B0AT1 (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 B0AT1 and its auxiliary peptidases as well as its transport function along the rat small intestine. Additionally, we tested its possible short- and long-term regulation by dietary proteins and amino acids. We showed by immunofluorescence that B0AT1, 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. Gastric emptying was delayed by intragastric application of an amino acid cocktail but we observed no acute dietary regulation of B0AT1 protein expression and L-isoleucine transport. Investigation of the chronic dietary regulation of B0AT1, ACE2 and CD13 by different diets revealed an increased B0AT1 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 B0AT1 but revealed a chronic dietary adaptation mainly restricted to the proximal segment of the small intestine.

Ubiquitin orchestrates proteasome dynamics between proliferation and quiescence in yeast.

Proteasomes are essential for protein degradation in proliferating cells. Little is known about proteasome functions in quiescent cells. In nondividing yeast, a eukaryotic model of quiescence, proteasomes are depleted from the nucleus and accumulate in motile cytosolic granules termed proteasome storage granules (PSGs). PSGs enhance resistance to genotoxic stress and confer fitness during aging. Upon exit from quiescence PSGs dissolve, and proteasomes are rapidly delivered into the nucleus. To identify key players in PSG organization, we performed high-throughput imaging of green fluorescent protein (GFP)-labeled proteasomes in the yeast null-mutant collection. Mutants with reduced levels of ubiquitin are impaired in PSG formation. Colocalization studies of PSGs with proteins of the yeast GFP collection, mass spectrometry, and direct stochastic optical reconstitution microscopy of cross-linked PSGs revealed that PSGs are densely packed with proteasomes and contain ubiquitin but no polyubiquitin chains. Our results provide insight into proteasome dynamics between proliferating and quiescent yeast in response to cellular requirements for ubiquitin-dependent degradation.

Human intestine luminal ACE2 and amino acid transporter expression increased by ACE-inhibitors.

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.

Blm10 facilitates nuclear import of proteasome core particles.

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 HEAT-like modules and is structurally related to the nuclear import receptor importin/karyopherin ß. 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.