Transport-dependent endocytosis and turnover of a uric acid-xanthine permease

Abstract

SummaryIn this work we unmask a novel downregulation mechanism of the uric acid/xanthine transporter UapA, the prototype member of the ubiquitous Nucleobase-Ascorbate Transporter family, directly related to its function. In the presence of substrates, UapA is endocytosed, sorted into the multivesicular body pathway and degraded in vacuoles. Substrateinduced endocytosis, unlike ammonium-induced turnover, is absolutely dependent on UapA activity and several lines of evidence showed that the signal for increased endocytosis is the actual translocation of substrates through the UapA protein. The use of several UapA functional mutants with altered kinetics and specificity has further shown that transportdependent UapA endocytosis occurs through a mechanism, which senses subtle conformational changes associated with the transport cycle. We also show that distinct mechanisms of UapA endocytosis necessitate ubiquitination of a single Lys residue (K572) by HulA Rsp5. Finally, we demonstrate that in the presence of substrates, non-functional UapA versions can be endocytosed in trans if expressed in the simultaneous presence of active UapA versions, even if the latter cannot be endocytosed themselves.

Substrate-Induced Ubiquitylation and Endocytosis of Yeast Amino Acid Permeases

“…This is reminiscent of what happens with the uric acid and xanthine permease (UapA) of A. nidulans, which undergoes ubiquitylation and endocytosis in response to Am but also to transport of its substrates (24). Substrate transport-induced endocytosis has also been demonstrated in the case of Fur4, the yeast uracil permease (23,25).…”

“…Still, it remains to be determined whether the signal inducing the endocytosis of these yAAPs is the internal accumulation of the transported amino acid (e.g., inhibiting the Npr1 kinase and thereby activating arrestin-like adaptors) or the catalytic process of amino acid transport itself. That transport catalysis might induce the Ub-dependent endocytosis of specific yAAPs is conceivable, as such a mechanism has been described for at least two fungal permeases, the uracil permease Fur4 (23) and the uric acid/xanthine permease UapA of Aspergillus nidulans (24). Recent work suggests that uracil-induced ubiquitylation of Fur4 is due to a conformational change causing disruption of an interaction between the membrane-proximal N-terminal tail and internal loops of the permease, thereby rendering N-terminal lysines more accessible to ubiquitylation by Rsp5 (25).…”

The Aspergillus nidulans Proline Permease as a Model for Understanding the Factors Determining Substrate Binding and Specificity of Fungal Amino Acid Transporters

“…Epifluorescence Microscopy-Samples were prepared as previously described (55,56). 20 mM L-proline was used for induction of prnB expression, at 25°C for 12-14 h. Samples were observed using a Plan-Apochromat ϫ100 1.40 NA oil immersion objective lens on an Axioplan 2 fluorescence microscope (Carl Zeiss, Inc.) with appropriate filters and the resulting images were acquired with a Zeiss-MRC5 digital camera using AxioVs40 V4.…”

“…Protein Extraction and In-gel Fluorescence-Membrane-enriched protein extracts were prepared as previously described (22,55), with the following modifications (58). Following membrane protein precipitation by centrifugation (1 h, 18,000 ϫ g, 4°C), the extraction buffer was removed and the pellet was resuspended in ice-cold In-gel Fluorescence Resuspension Buffer (IFRB: 50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 10% glycerol) freshly supplemented with protease inhibitor mixture (Sigma, P8215, 1:500) and 1 mM PMSF.…”

Eisosome Organization in the Filamentous AscomyceteAspergillus nidulans

“…Load-and-chase experiments have shown that when the dye FM4-64 is endocytosed, it first appears in cortical punctate structures (36). Active research concerning membrane proteins (see reference 12 for a review), determination and maintenance of polarity during development (including the possible involvement of sphingolipids [23], which may be involved in signaling eisosomal protein phosphorylation in S. cerevisiae), and ongoing recent work on endocytosis (1,5,6,16,36,40) make A. nidulans an obvious model within the Pezizomycotina with which to study eisosomal presence, structure, and function. A description of the presence and fate of these organelles during asexual development is presented below.…”