SummaryThe accurate formation of synaptic vesicles (SVs) and incorporation of their protein cargo during endocytosis is critical for the maintenance of neurotransmission. During intense neuronal activity, a transient and acute accumulation of SV cargo occurs at the plasma membrane. Activity-dependent bulk endocytosis (ADBE) is the dominant SV endocytosis mode under these conditions; however, it is currently unknown how ADBE mediates cargo retrieval. We examined the retrieval of different SV cargo molecules during intense stimulation using a series of genetically encoded pH-sensitive reporters in neuronal cultures. The retrieval of only one reporter, VAMP4-pHluorin, was perturbed by inhibiting ADBE. This selective recovery was confirmed by the enrichment of endogenous VAMP4 in purified bulk endosomes formed by ADBE. VAMP4 was also essential for ADBE, with a cytoplasmic di-leucine motif being critical for this role. Therefore, VAMP4 is the first identified ADBE cargo and is essential for this endocytosis mode to proceed.
SummaryWe investigated the role of all arrestin-like proteins of Aspergillus nidulans in respect to growth, morphology, sensitivity to drugs and specifically for the endocytosis and turnover of the uric acid-xanthine transporter UapA. A single arrestin-like protein, ArtA, is essential for HulA Rsp5-dependent ubiquitination and endocytosis of UapA in response to ammonium or substrates. Mutational analysis showed that residues 545-563 of the UapA C-terminal region are required for efficient UapA endocytosis, whereas the N-terminal region (residues 2-123) and both PPxY motives are essential for ArtA function. We further show that ArtA undergoes HulA-dependent ubiquitination at residue Lys-343 and that this modification is critical for UapA ubiquitination and endocytosis. Lastly, we show that ArtA is essential for vacuolar turnover of transporters specific for purines (AzgA) or L-proline (PrnB), but not for an aspartate/glutamate transporter (AgtA). Our results are discussed within the frame of recently proposed mechanisms on how arrestin-like proteins are activated and recruited for ubiquitination of transporters in response to broad range signals, but also put the basis for understanding how arrestin-like proteins, such as ArtA, regulate the turnover of a specific transporter in the presence of its substrates.
SignificanceThe maintenance of neurotransmission by synaptic vesicle (SV) recycling is critical to brain function. The dominant SV recycling mode during intense activity is activity-dependent bulk endocytosis (ADBE), suggesting it will perform a pivotal role in neurotransmission. However, the role of ADBE is still undetermined, due to the absence of identified molecules specific for this process. The determination of the bulk endosome proteome (a key ADBE organelle) revealed that it has a unique molecular signature and identified a role for Rab11 in presynaptic function. This work provides the molecular inventory of ADBE, a resource that will be of significant value to researchers wishing to modulate neurotransmission during intense neuronal activity in both health and disease.
Type 2 diabetes (T2D) is a complex metabolic disease associated with obesity, insulin resistance and hypoinsulinemia due to pancreatic β-cell dysfunction. Reduced mitochondrial function is thought to be central to β-cell dysfunction. Mitochondrial dysfunction and reduced insulin secretion are also observed in β-cells of humans with the most common human genetic disorder, Down syndrome (DS, Trisomy 21). To identify regions of chromosome 21 that may be associated with perturbed glucose homeostasis we profiled the glycaemic status of different DS mouse models. The Ts65Dn and Dp16 DS mouse lines were hyperglycemic, while Tc1 and Ts1Rhr mice were not, providing us with a region of chromosome 21 containing genes that cause hyperglycemia. We then examined whether any of these genes were upregulated in a set of ~5,000 gene expression changes we had identified in a large gene expression analysis of human T2D β-cells. This approach produced a single gene, RCAN1, as a candidate gene linking hyperglycemia and functional changes in T2D β-cells. Further investigations demonstrated that RCAN1 methylation is reduced in human T2D islets at multiple sites, correlating with increased expression. RCAN1 protein expression was also increased in db/db mouse islets and in human and mouse islets exposed to high glucose. Mice overexpressing RCAN1 had reduced in vivo glucose-stimulated insulin secretion and their β-cells displayed mitochondrial dysfunction including hyperpolarised membrane potential, reduced oxidative phosphorylation and low ATP production. This lack of β-cell ATP had functional consequences by negatively affecting both glucose-stimulated membrane depolarisation and ATP-dependent insulin granule exocytosis. Thus, from amongst the myriad of gene expression changes occurring in T2D β-cells where we had little knowledge of which changes cause β-cell dysfunction, we applied a trisomy 21 screening approach which linked RCAN1 to β-cell mitochondrial dysfunction in T2D.
Central nerve terminals contain a small number of synaptic vesicles (SVs) that must sustain the fidelity of neurotransmission across a wide range of stimulation intensities. For this to be achieved, nerve terminals integrate a number of complementary endocytosis modes whose activation spans the breadth of these neuronal stimulation patterns. Two such modes are ultrafast endocytosis and activity-dependent bulk endocytosis, which are triggered by stimuli at either end of the physiological range. Both endocytosis modes generate endosomes directly from the nerve terminal plasma membrane, before the subsequent production of SVs from these structures. This review will discuss the current knowledge relating to the molecular mechanisms involved in the generation of SVs from nerve terminal endosomes, how this relates to other mechanisms of SV production and the functional role of such SVs.
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