Role of the mammalian retromer in sorting of the cation-independent mannose 6-phosphate receptor

The cation-independent mannose 6-phosphate receptor (CI-MPR) mediates sorting of lysosomal hydrolase precursors from the TGN to endosomes. After releasing the hydrolase precursors into the endosomal lumen, the unoccupied receptor returns to the TGN for further rounds of sorting. Here, we show that the mammalian retromer complex participates in this retrieval pathway. The hVps35 subunit of retromer interacts with the cytosolic domain of the CI-MPR. This interaction probably occurs in an endosomal compartment, where most of the retromer is localized. In particular, retromer is associated with tubular–vesicular profiles that emanate from early endosomes or from intermediates in the maturation from early to late endosomes. Depletion of retromer by RNA interference increases the lysosomal turnover of the CI-MPR, decreases cellular levels of lysosomal hydrolases, and causes swelling of lysosomes. These observations indicate that retromer prevents the delivery of the CI-MPR to lysosomes, probably by sequestration into endosome-derived tubules from where the receptor returns to the TGN

Simple and informative: applying a basic Anthophila monitoring scheme in a simplified insular ecosystem

The decline of pollinators and the consequent decay of pollination services call for the establishment of monitoring schemes for several groups of pollinators. For Anthophila (Hymenoptera), the design of monitoring schemes is still under development. The main difficulties lie in combining a reliable but field-feasible taxonomic identification with the collection of informative data about the consistency and functional role of pollinator populations. Here we report on the application of the Italian monitoring scheme for pollinators recently defined by ISPRA and the University of Turin in agreement with the European Pollinators Monitoring Scheme on the small island of Giannutri (Tuscany), a simplified insular ecosystem with a virtually unknown pollinator community. This island has recently experienced a drastic change in its bee community, as since 2018 honey bee (Apis mellifera L.) hives are regularly moved every year to the island for breeding purposes. In the spring 2021 we established six 250 m long fixed transects and performed a total of 48 surveys (8 for each transect), recording more than 2300 observations of 9 Anthophila bee taxa and the flowers they visited. By using generalised additive mixed models, we showed that the monitoring protocol has a good potential for monitoring Anthophila, as we could verify several expected relationships between Anthophila abundance and abiotic factors (season, hour of the day, distance from the apiary) and biotic factors (abundance of flower resources). More importantly, we verified that A. mellifera represents by far the most frequent Anthophila taxon. Our data do not show evidence for spatial partition between A. mellifera and the other most frequent taxa (Bombus terrestris L. and Anthophora spp.). The visit network based on transect observations also showed that these taxa largely overlapped in terms of visits to flower resources. Overall, our data showed that the monitoring protocol allows gathering informative data about Anthophila taxa abundance, interactions and flower-visits. Moreover, the spatial and flower-visit overlap suggest potential for competition between honey bees and wild pollinators, with a potential consequent resource depletion for the latter. While this hypothesis could only be assessed by a long-term monitoring and ad hoc honey bee removal experiments, our data show that this basic monitoring protocol produces rapid and valuable information about Anthophila community and dynamics

Recognition of dileucine-based sorting signals from HIV-1 Nef and LIMP-II by the AP-1 γ–σ1 and AP-3 δ–σ3 hemicomplexes

The sorting of transmembrane proteins to endosomes and lysosomes is mediated by signals present in the cytosolic tails of the proteins. A subset of these signals conform to the [DE]XXXL[LI] consensus motif and mediate sorting via interactions with heterotetrameric adaptor protein (AP) complexes. However, the identity of the AP subunits that recognize these signals remains controversial. We have used a yeast three-hybrid assay to demonstrate that [DE]XXXL[LI]-type signals from the human immunodeficiency virus negative factor protein and the lysosomal integral membrane protein II interact with combinations of the γ and σ1 subunits of AP-1 and the δ and σ3 subunits of AP-3, but not the analogous combinations of AP-2 and AP-4 subunits. The sequence requirements for these interactions are similar to those for binding to the whole AP complexes in vitro and for function of the signals in vivo. These observations reveal a novel mode of recognition of sorting signals involving the γ/δ and σ subunits of AP-1 and AP-3

Crystallographic and Functional Analysis of the ESCRT-I /HIV-1 Gag PTAP Interaction

SummaryBudding of HIV-1 requires the binding of the PTAP late domain of the Gag p6 protein to the UEV domain of the TSG101 subunit of ESCRT-I. The normal function of this motif in cells is in receptor downregulation. Here, we report the 1.4–1.6 Å structures of the human TSG101 UEV domain alone and with wild-type and mutant HIV-1 PTAP and Hrs PSAP nonapeptides. The hydroxyl of the Thr or Ser residue in the P(S/T)AP motif hydrogen bonds with the main chain of Asn69. Mutation of the Asn to Pro, blocking the main-chain amide, abrogates PTAP motif binding in vitro and blocks budding of HIV-1 from cells. N69P and other PTAP binding-deficient alleles of TSG101 did not rescue HIV-1 budding. However, the mutant alleles did rescue downregulation of endogenous EGF receptor. This demonstrates that the PSAP motif is not rate determining in EGF receptor downregulation under normal conditions

The Sec1/Munc18 protein Vps45 regulates cellular levels of its SNARE binding partners Tlg2 and Snc2 in Saccharomyces cerevisiae

Intracellular membrane trafficking pathways must be tightly regulated to ensure proper functioning of all eukaryotic cells. Central to membrane trafficking is the formation of specific SNARE (soluble N-ethylmeleimide-sensitive factor attachment protein receptor) complexes between proteins on opposing lipid bilayers. The Sec1/Munc18 (SM) family of proteins play an essential role in SNARE-mediated membrane fusion, and like the SNAREs are conserved through evolution from yeast to humans. The SM protein Vps45 is required for the formation of yeast endosomal SNARE complexes and is thus essential for traffic through the endosomal system. Here we report that, in addition to its role in regulating SNARE complex assembly, Vps45 regulates cellular levels of its SNARE binding partners: the syntaxin Tlg2 and the v-SNARE Snc2: Cells lacking Vps45 have reduced cellular levels of Tlg2 and Snc2; and elevation of Vps45 levels results in concomitant increases in the levels of both Tlg2 and Snc2. As well as regulating traffic through the endosomal system, the Snc v-SNAREs are also required for exocytosis. Unlike most vps mutants, cells lacking Vps45 display multiple growth phenotypes. Here we report that these can be reversed by selectively restoring Snc2 levels in vps45 mutant cells. Our data indicate that as well as functioning as part of the machinery that controls SNARE complex assembly, Vps45 also plays a key role in determining the levels of its cognate SNARE proteins; another key factor in regulation of membrane traffic