We have recently shown that two proteins related to two of the adaptor subunits of clathrincoated vesicles, p47 (mu3) and beta-NAP (beta3B), are part of an adaptor-like complex not associated with clathrin (Simpson, F., N.A. Bright, M.A. West, L.S. Newman, R.B. Darnell, and M.S. Robinson, 1996. J. Cell Biol. 133:749-760). In the present study we have searched the EST database and have identified, cloned, and sequenced a ubiquitously expressed homologue of beta-NAP, beta3A, as well as homologues of the alpha/gamma and sigma adaptor subunits, delta and sigma3, which are also ubiquitously expressed. Antibodies raised against recombinant delta and sigma3 show that they are the other two subunits of the adaptor-like complex. We are calling this complex AP-3, a name that has also been used for the neuronalspecific phosphoprotein AP180, but we feel that it is a more appropriate designation for an adaptor-related heterotetramer. Immunofluorescence using anti-delta antibodies reveals that the AP-3 complex is associated with the Golgi region of the cell as well as with more peripheral structures. These peripheral structures show only limited colocalization with endosomal markers and may correspond to a postTGN biosynthetic compartment. The delta subunit is closely related to the protein product of the Drosophila garnet gene, which when mutated results in reduced pigmentation of the eyes and other tissues. Because pigment granules are believed to be similar to lysosomes, this suggests either that the AP-3 complex may be directly involved in trafficking to lysosomes or alternatively that it may be involved in another pathway, but that missorting in that pathway may indirectly lead to defects in pigment granules.

The trans-Golgi network (TGN) is the main secretory pathway sorting station, where cargoes are packed into appropriate transport vesicles targeted to specific destinations. Exomer is a cargo adaptor necessary for direct transport of a subset of cargoes from the TGN to the plasma membrane in yeast. Here, we show that unlike classical adaptor complexes, exomer is not recruited en bloc to the TGN, but rather assembles through a stepwise pathway, in which first the scaffold protein Chs5 and then the cargo-binding units, the ChAPs, are recruited. Although all ChAPs are able to assemble functional exomer complexes, they do so with different efficiencies. The mutual relationship between ChAPs varies from cooperation to competition depending on their expression levels and affinities to Chs5 allowing an optimized and efficient cargo transport. The multifactorial assembly pathway results in an exquisitely fine-tuned adaptor complex, enabling the cell to quickly respond and adapt to changes such as stress.

The adaptor protein-2 sigma subunit (AP2σ), encoded by AP2S1, forms a heterotetrameric complex, with AP2α, AP2β, and AP2μ subunits, that is pivotal for clathrin-mediated endocytosis, and AP2σ loss-of-function mutations impair internalization of the calcium-sensing receptor (CaSR), a G-protein-coupled receptor, and cause familial hypocalciuric hypercalcemia type-3 (FHH3). Mice with AP2σ mutations that would facilitate investigations of the in vivo role of AP2σ, are not available, and we therefore embarked on establishing such mice. We screened >10,000 mice treated with the mutagen N-ethyl-N-nitrosourea (ENU) for Ap2s1 mutations and identified 5 Ap2s1 variants, comprising 2 missense (Tyr20Asn and Ile123Asn) and 3 intronic base substitutions, one of which altered the invariant donor splice site dinucleotide gt to gc. Three-dimensional modeling and cellular expression of the missense Ap2s1 variants did not reveal them to alter AP2σ structure or CaSR-mediated signaling, but investigation of the donor splice site variant revealed it to result in an in-frame deletion of 17 evolutionarily conserved amino acids (del17) that formed part of the AP2σ α1-helix, α1-β3 loop, and β3 strand. Heterozygous mutant mice (Ap2s1+/del17 ) were therefore established, and these had AP2σ haplosufficiency but were viable with normal appearance and growth. Ap2s1+/del17 mice, when compared with Ap2s1+/+ mice, also had normal plasma concentrations of calcium, phosphate, magnesium, creatinine, urea, sodium, potassium, and alkaline phosphatase activity; normal urinary fractional excretion of calcium, phosphate, sodium, and potassium; and normal plasma parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D (1,25(OH)2) concentrations. However, homozygous Ap2s1del17/del17 mice were non-viable and died between embryonic days 3.5 and 9.5 (E3.5-9.5), thereby indicating that AP2σ likely has important roles at the embryonic patterning stages and organogenesis of the heart, thyroid, liver, gut, lungs, pancreas, and neural systems. Thus, our studies have established a mutant mouse model that is haplosufficient for AP2σ.

Mutations of the sigma subunit of the heterotetrameric adaptor-related protein complex 2 (AP2σ) impair signalling of the calcium-sensing receptor (CaSR), and cause familial hypocalciuric hypercalcaemia type 3 (FHH3). To date, FHH3-associated AP2σ mutations have only been identified at one residue, Arg15. We hypothesized that additional rare AP2σ variants may also be associated with altered CaSR function and hypercalcaemia, and sought for these by analysing >111 995 exomes (>60 706 from ExAc and dbSNP, and 51 289 from the Geisinger Health System-Regeneron DiscovEHR dataset, which also contains clinical data). This identified 11 individuals to have 9 non-synonymous AP2σ variants (Arg3His, Arg15His (x3), Ala44Thr, Phe52Tyr, Arg61His, Thr112Met, Met117Ile, Glu122Gly and Glu142Lys) with 3 of the 4 individuals who had Arg15His and Met117Ile AP2σ variants having mild hypercalcaemia, thereby indicating a prevalence of FHH3-associated AP2σ mutations of ∼7.8 per 100 000 individuals. Structural modelling of the novel eight AP2σ variants (Arg3His, Ala44Thr, Phe52Tyr, Arg61His, Thr112Met, Met117Ile, Glu122Gly and Glu142Lys) predicted that the Arg3His, Thr112Met, Glu122Gly and Glu142Lys AP2σ variants would disrupt polar contacts within the AP2σ subunit or affect the interface between the AP2σ and AP2α subunits. Functional analyses of all eight AP2σ variants in CaSR-expressing cells demonstrated that the Thr112Met, Met117Ile and Glu142Lys variants, located in the AP2σ α4-α5 helical region that forms an interface with AP2α, impaired CaSR-mediated intracellular calcium (Cai2+) signalling, consistent with a loss of function, and this was rectified by treatment with the CaSR positive allosteric modulator cinacalcet. Thus, our studies demonstrate another potential class of FHH3-causing AP2σ mutations located at the AP2σ-AP2α interface.

Previously, we reported that the mediation of Newcastle disease virus (NDV) pathogenicity by the 524YLMY527 motif depends mainly on the regulation of F protein transport to the cell surface. The virus and host determinants that govern this intracellular trafficking remain unknown. Here, we confirmed that host adaptor protein (AP) complexes are involved in NDV infection using small interfering RNA. The transport of viral F protein to the cell surface depends on host transport proteins. We observed that the trends for host expression of AP complexes AP1M1 and AP2M1 were similar to those of mutated F proteins, especially in the membrane protein. NDV F protein interacted with AP1M1 and AP2M1, and the YLMY motif influenced this interaction. Knockdown of AP1M1 or AP2M1 suppressed the intracellular and extracellular virus titre of mutated-YLMY-motif NDVs, especially rSG10*-F/Y527A and rSG10*-F/Y524AY527A, to varying degrees. Therefore, the YLMY motif regulates AP-mediated viral F protein transportation from the cytoplasm to the cell surface and subsequently affects viral titer. We further found that the YLMY-motif mutants were differently associated with the process of AAK1 and GAK kinase-mediated AP - viral F protein interaction. These data demonstrate that the essential YLMY motif located in the NDV F protein cytoplasmic tail recruits AP to direct the F protein to the cell surface, which is necessary for its ability to affect virus budding. This study provides support for a deeper understanding of virus and host determinants that facilitate virus trafficking, which can be exploited in the design of novel antiviral therapies.

LZTFL1 participates in immune synapse formation, ciliogenesis, and the localization of ciliary proteins, and knockout of LZTFL1 induces abnormal distribution of heterotetrameric adaptor protein complex-1 (AP-1) in the Lztfl1-knockout mouse photoreceptor cells, suggesting that LZTFL1 is involved in intracellular transport. Here, we demonstrate that in vitro LZTFL1 directly binds to AP-1 and AP-2 and coimmunoprecipitates AP-1 and AP-2 from cell lysates. DxxFxxLxxxR motif of LZTFL1 is essential for these bindings, suggesting LZTFL1 has roles in AP-1 and AP-2-mediated protein trafficking. Since AP-1 and AP-2 are known to be involved in transferrin receptor 1 (TfR1) trafficking, the effect of LZTFL1 on TfR1 recycling was analyzed. TfR1, AP-1 and LZTFL1 from cell lysates could be coimmunoprecipitated. However, pull-down results indicate there is no direct interaction between TfR1 and LZTFL1, suggesting that LZTFL1 interaction with TfR1 is indirect through AP-1. We report the colocalization of LZTFL1 and AP-1, AP-1 and TfR1 as well as LZTFL1 and TfR1 in the perinuclear region (PNR) and the cytoplasm, suggesting a potential complex between LZTFL1, AP-1 and TfR1. The results from the disruption of adaptin recruitment with brefeldin A treatment suggested ADP-ribosylation factor-dependent localization of LZFL1 and AP-1 in the PNR. Knockdown of AP-1 reduces the level of LZTFL1 in the PNR, suggesting that AP-1 plays a role in LZTFL1 trafficking. Knockout of LZTFL1 reduces the cell surface level and the rate of internalization of TfR1, leading to a decrease of transferrin uptake, efflux, and internalization. However, knockout of LZTFL1 did not affect the cell surface levels of epidermal growth factor receptor and cation-independent mannose 6-phosphate receptor, indicating that LZTFL1 specifically regulates the cell surface level of TfR1. These data support a novel role of LZTFL1 in regulating the cell surface TfR1 level by interacting with AP-1 and AP-2.

Multimeric adaptors are broadly involved in vesicle-mediated membrane trafficking. AP2 adaptor, in particular, plays a central role in clathrin-mediated endocytosis (CME) by recruiting cargo and clathrin to endocytic sites. It is generally thought that trafficking adaptors such as AP2 adaptor assemble spontaneously. In this work, however, we discovered that AP2 adaptor assembly is an ordered process controlled by alpha and gamma adaptin binding protein (AAGAB), an uncharacterized factor identified in our genome-wide genetic screen of CME. AAGAB guides the sequential association of AP2 subunits and stabilizes assembly intermediates. Without the assistance of AAGAB, AP2 subunits fail to form the adaptor complex, leading to their degradation. The function of AAGAB is abrogated by a mutation that causes punctate palmoplantar keratoderma type 1 (PPKP1), a human skin disease. Since other multimeric trafficking adaptors operate in an analogous manner to AP2 adaptor, their assembly likely involves a similar regulatory mechanism.

The human herpesvirus-7 (HHV-7) U21 gene product binds to class I major histocompatibility complex (MHC) molecules and reroutes them to a lysosomal compartment. Trafficking of integral membrane proteins to lysosomes is mediated through cytoplasmic sorting signals that recruit heterotetrameric clathrin adaptor protein (AP) complexes, which in turn mediate protein sorting in post-Golgi vesicular transport. Since U21 can mediate rerouting of class I molecules to lysosomes even when lacking its cytoplasmic tail, we hypothesize the existence of a cellular protein that contains the lysosomal sorting information required to escort class I molecules to the lysosomal compartment. If such a protein exists, we expect that it might recruit clathrin adaptor protein complexes as a means of lysosomal sorting. Here we describe experiments demonstrating that the μ adaptins from AP-1 and AP-3 are involved in U21-mediated trafficking of class I molecules to lysosomes. These experiments support the idea that a cellular protein(s) is necessary for U21-mediated lysosomal sorting of class I molecules. We also examine the impact of transient versus chronic knockdown of these adaptor protein complexes, and show that the few remaining μ subunits in the cells are eventually able to reroute class I molecules to lysosomes.

The high reproductive rates of insects contribute significantly to their ability to act as vectors of a variety of vector-borne diseases. Therefore, it is strategically critical to find molecular targets with biotechnological potential through the functional study of genes essential for insect reproduction. The ubiquitin-proteasome system is a vital degradative pathway that contributes to the maintenance of regular eukaryotic cell proteostasis. This mechanism involves the action of enzymes to covalently link ubiquitin to proteins that are meant to be delivered to the 26S proteasome and broken down. The 26S proteasome is a large protease complex (including the 20S and 19S subcomplexes) that binds, deubiquitylates, unfolds, and degrades its substrates. Here, we used bioinformatics to identify the genes that encode the seven α and β subunits of the 20S proteasome in the genome of R. prolixus and learned that those transcripts are accumulated into mature oocytes. To access proteasome function during oogenesis, we conducted RNAi functional tests employing one of the 20S proteasome subunits (Prosα6) as a tool to suppress 20S proteasomal activity. We found that Prosα6 silencing resulted in no changes in TAG buildup in the fat body and unaffected availability of yolk proteins in the hemolymph of vitellogenic females. Despite this, the silencing of Prosα6 culminated in the impairment of oocyte maturation at the early stages of oogenesis. Overall, we discovered that proteasome activity is especially important for the signals that initiate oogenesis in R. prolixus and discuss in what manner further investigations on the regulation of proteasome assembly and activity might contribute to the unraveling of oogenesis molecular mechanisms and oocyte maturation in this vector.

IL-17 is the founding member of a family of cytokines and receptors with unique structures and signaling properties. IL-17 is the signature cytokine of Th17 cells, a relatively new T cell population that promotes inflammation in settings of infection and autoimmunity. Despite advances in understanding Th17 cells, mechanisms of IL-17-mediated signal transduction are less well defined. IL-17 signaling requires contributions from two receptor subunits, IL-17RA and IL-17RC. Mutants of IL-17RC lacking the cytoplasmic domain are nonfunctional, indicating that IL-17RC provides essential but poorly understood signaling contributions to IL-17-mediated signaling. To better understand the role of IL-17RC in signaling, we performed a yeast 2-hybrid screen to identify novel proteins associated with the IL-17RC cytoplasmic tail. One of the most frequent candidates was the anaphase promoting complex protein 7 (APC7 or AnapC7), which interacted with both IL-17RC and IL-17RA. Knockdown of AnapC7 by siRNA silencing exerted no detectable impact on IL-17 signaling. However, AnapC5, which associates with AnapC7, was also able to bind IL-17RA and IL-17RC. Moreover, AnapC5 silencing enhanced IL-17-induced gene expression, suggesting an inhibitory activity. Strikingly, AnapC5 also associated with A20 (TNFAIP3), a recently-identified negative feedback regulator of IL-17 signal transduction. IL-17 signaling was not impacted by knockdown of Itch or TAXBP1, scaffolding proteins that mediate A20 inhibition in the TNFα and IL-1 signaling pathways. These data suggest a model in which AnapC5, rather than TAX1BP1 and Itch, is a novel adaptor and negative regulator of IL-17 signaling pathways.

Coats define the composition of carriers budding from organelles. In addition, coats interact with membrane tethers required for vesicular fusion. The yeast AP-3 (Adaptor Protein Complex 3) coat and the class C Vps/HOPS (HOmotypic fusion and Protein Sorting) tether follow this model as their interaction occurs at the carrier fusion step. Here we show that mammalian Vps class C/HOPS subunits and clathrin interact and that acute perturbation of clathrin function disrupts the endosomal distribution of Vps class C/HOPS tethers in HEK293T and polarized neuronal cells. Vps class C/HOPS subunits and clathrin exist in complex with either AP-3 or hepatocyte growth factor receptor substrate (Hrs). Moreover, Vps class C/HOPS proteins cofractionate with clathrin-coated vesicles, which are devoid of Hrs. Expression of FK506 binding protein (FKBP)-clathrin light chain chimeras, to inhibit clathrin membrane association dynamics, increased Vps class C/HOPS subunit content in rab5 endosomal compartments. Additionally, Vps class C/HOPS subunits were concentrated at tips of neuronal processes, and their delivery was impaired by expression of FKBP-clathrin chimeras and AP20187 incubation. These data support a model in which Vps class C/HOPS subunits incorporate into clathrin-coated endosomal domains and carriers in mammalian cells. We propose that vesicular (AP-3) and nonvesicular (Hrs) clathrin mechanisms segregate class C Vps/HOPS tethers to organelles and domains of mammalian cells bearing complex architectures.

Cilia are found on most eukaryotic cell types, serving motility, environment sensing, and signaling (cell-cell) functions, and defects cause genetic diseases (ciliopathies), affecting the development of many tissues [1]. Cilia are built by intraflagellar transport (IFT), a bidirectional microtubule-based motility driven by kinesin-2 anterograde (toward ciliary tip) and IFT-dynein retrograde (toward ciliary base) motors together with IFT-A and IFT-B cargo adaptor complexes that control retrograde and anterograde IFT, respectively [2]. Ciliary composition is also facilitated by the transition zone (TZ) at the ciliary base and the associated Meckel-Gruber syndrome (MKS) and nephronophthisis (NPHP) modules that establish protein diffusion barriers and regulate cilium structure [3]. Although the molecular architecture of the IFT machine is emerging [2], how individual components contribute to cilium subtype formation and IFT remains relatively unexplored, especially in vivo. In addition, little is known about functional interactions between IFT and TZ modules. Here, in Caenorhabditis elegans (roundworms), we identify cell-type-specific mechanisms by which IFT-A sculpts the structures of discrete ciliary subtypes and regulates IFT. We also uncover differential roles for IFT-A subunits in controlling the TZ restriction of MKS module components and ciliary exclusion (gating) of periciliary membrane proteins, with IFT-140 controlling their ciliary entry and IFT-43/121/139 controlling their ciliary removal. Furthermore, we determine that IFT-A and MKS module components synergistically interact to determine cilium structure. Overall, this work provides insight into the functional architecture of a metazoan IFT-A complex in different cell types and uncovers new relationships between ciliopathy-associated IFT-A and TZ modules.

Kv4 α-subunits exist as ternary complexes (TC) with potassium channel interacting proteins (KChIP) and dipeptidyl peptidase-like proteins (DPLP); multiple ancillary proteins also interact with the α-subunits throughout the channel's lifetime. Dynamic regulation of Kv4.2 protein interactions adapts the transient potassium current, IA, mediated by Kv4 α-subunits. Small ubiquitin-like modifier (SUMO) is an 11 kD peptide post-translationally added to lysine (K) residues to regulate protein-protein interactions. We previously demonstrated that when expressed in human embryonic kidney (HEK) cells, Kv4.2 can be SUMOylated at two K residues, K437 and K579. SUMOylation at K437 increased surface expression of electrically silent channels while SUMOylation at K579 reduced IA maximal conductance (Gmax) without altering surface expression. KChIP and DPLP subunits are known to modify the pattern of Kv4.2 post-translational decorations and/or their effects. In this study, co-expressing Kv4.2 with KChIP2a and DPP10c altered the effects of enhanced Kv4.2 SUMOylation. First, the effect of enhanced SUMOylation was the same for a TC containing either the wild-type Kv4.2 or the mutant K437R Kv4.2, suggesting that either the experimental manipulation no longer enhanced K437 SUMOylation or K437 SUMOylation no longer influenced Kv4.2 surface expression. Second, instead of decreasing IA Gmax, enhanced SUMOylation at K579 now produced a significant ∼37-70% increase in IA maximum conductance (Gmax) and a significant ∼30-50% increase in Kv4.2g surface expression that was accompanied by a 65% reduction in TC internalization. Blocking clathrin-mediated endocytosis (CME) in HEK cells expressing the Kv4.2 TC mimicked and occluded the effect of SUMO on IA Gmax; however, the amount of Kv4.2 associated with the major adaptor for constitutive CME, adaptor protein 2 (AP2), was not SUMO dependent. Thus, SUMOylation reduced Kv4.2 internalization by acting downstream of Kv4.2 recruitment into clathrin-coated pits. In sum, the two major findings of this study are: SUMOylation of Kv4.2 at K579 regulates TC internalization most likely by promoting channel recycling. Additionally, there is a reciprocity between Kv4.2 SUMOylation and the Kv4.2 interactome such that SUMOylation regulates the interactome and the interactome influences the pattern and effect of SUMOylation.

Curli play critical roles in biofilm formation, host cell adhesion, and colonization of inert surfaces in many Enterobacteriaceae. In Escherichia coli, curli biogenesis requires 7 curli-specific gene (csg) products-CsgA through G-working in concert. Of them, CsgG and CsgF are 2 outer membrane (OM)-localized components that consists of the core apparatus for secretion and assembly of curli structural subunits, CsgB and CsgA. Here, we report the cryogenic electron microscopy (cryo-EM) structure of CsgG in complex with CsgF from E. coli. The structure reveals that CsgF forms a stable complex with CsgG via a 1:1 stoichiometry by lining the upper lumen of the nonameric CsgG channel via its N-terminal 27 residues, forming a funnel-like entity plugged in the CsgG channel and creating a unique secretion channel with 2 constriction regions, consistent with the recently reported structure of the CsgG-CsgF complex. Functional studies indicate that export of CsgF to the cell surface requires the CsgG channel, and CsgF not only functions as an adaptor that bridges CsgB with CsgG but also may play important roles in controlling the rates of translocation and/or polymerization for curli structural subunits. Importantly, we found that a series of CsgF-derived peptides are able to efficiently inhibit curli production to E. coli when administrated exogenously, highlighting a potential strategy to interfere biofilm formation in E. coli strains.

Many long noncoding RNAs (lncRNAs) are unstable and rapidly degraded in the nucleus by the nuclear exosome. An exosome adaptor complex called NEXT (nuclear exosome targeting) functions to facilitate turnover of some of these lncRNAs. Here we show that knockdown of one NEXT subunit, Mtr4, but neither of the other two subunits, resulted in accumulation of two types of lncRNAs: prematurely terminated RNAs (ptRNAs) and upstream antisense RNAs (uaRNAs). This suggested a NEXT-independent Mtr4 function, and, consistent with this, we isolated a distinct complex containing Mtr4 and the zinc finger protein ZFC3H1. Strikingly, knockdown of either protein not only increased pt/uaRNA levels but also led to their accumulation in the cytoplasm. Furthermore, all pt/uaRNAs examined associated with active ribosomes, but, paradoxically, this correlated with a global reduction in heavy polysomes and overall repression of translation. Our findings highlight a critical role for Mtr4/ZFC3H1 in nuclear surveillance of naturally unstable lncRNAs to prevent their accumulation, transport to the cytoplasm, and resultant disruption of protein synthesis.

The homohexameric p97 complex, composed of Cdc48 subunits in yeast, is a crucial component of protein quality control pathways including ER-associated degradation. The complex acts to segregate protein complexes in an ATP-dependent manner, requiring the engagement of cofactor proteins that determine substrate specificity. The function of different Cdc48 cofactors and how they are regulated remains relatively poorly understood. In this study, we assess the phosphorylation of Cdc48 adaptor proteins, revealing a unique and distinctive phosphorylation pattern of Shp1/p47 that changed in response to TORC1 inhibition. Site-directed mutagenesis confirmed that this pattern corresponded to phosphorylation at residues S108 and S315 of Shp1, with the double-phosphorylated form becoming predominant upon TORC1 inhibition, ER-stress, and oxidative stress. Finally, we assessed candidate kinases and phosphatases responsible for Shp1 phosphorylation and identified two regulators. We found that cells lacking the kinase Mpk1/Slt2 show reduced Shp1 phosphorylation, whereas impaired PP1 phosphatase catalytic subunit (Glc7) activity resulted in increased Shp1 phosphorylation. Overall, these findings identify a phosphoregulation of Shp1 at multiple sites by Mpk1 kinase and PP1 phosphatase upon various stresses.

E3 cullin-RING ubiquitin ligase (CRL) complexes recognize specific substrates and are activated by covalent modification with ubiquitin-like Nedd8. Deneddylation inactivates CRLs and allows Cand1/A to bind and exchange substrate recognition subunits. Human as well as most fungi possess a single gene for the receptor exchange factor Cand1, which is split and rearranged in aspergilli into two genes for separate proteins. Aspergillus nidulans CandA-N blocks the neddylation site, and CandA-C inhibits the interaction to the adaptor/substrate receptor subunits similar to the respective N-terminal and C-terminal parts of single Cand1. The pathogen Aspergillus fumigatus and related species express a CandA-C with a 190-amino-acid N-terminal extension domain encoded by an additional exon. This extension corresponds in most aspergilli, including A. nidulans, to a gene directly upstream of candA-C encoding a 20-kDa protein without human counterpart. This protein was named CandA-C1, because it is also required for the cellular deneddylation/neddylation cycle and can form a trimeric nuclear complex with CandA-C and CandA-N. CandA-C and CandA-N are required for asexual and sexual development and control a distinct secondary metabolism. CandA-C1 and the corresponding domain of A. fumigatus control spore germination, vegetative growth, and the repression of additional secondary metabolites. This suggests that the dissection of the conserved Cand1-encoding gene within the genome of aspergilli was possible because it allowed the integration of a fungus-specific protein required for growth into the CandA complex in two different gene set versions, which might provide an advantage in evolution.IMPORTANCEAspergillus species are important for biotechnological applications, like the production of citric acid or antibacterial agents. Aspergilli can cause food contamination or invasive aspergillosis to immunocompromised humans or animals. Specific treatment is difficult due to limited drug targets and emerging resistances. The CandA complex regulates, as a receptor exchange factor, the activity and substrate variability of the ubiquitin labeling machinery for 26S proteasome-mediated protein degradation. Only Aspergillus species encode at least two proteins that form a CandA complex. This study shows that Aspergillus species had to integrate a third component into the CandA receptor exchange factor complex that is unique to aspergilli and required for vegetative growth, sexual reproduction, and activation of the ubiquitin labeling machinery. These features have interesting implications for the evolution of protein complexes and could make CandA-C1 an interesting candidate for target-specific drug design to control fungal growth without affecting the human ubiquitin-proteasome system.

TMEM39A, encoding an ER-localized transmembrane protein, is a susceptibility locus for multiple autoimmune diseases. The molecular function of TMEM39A remains completely unknown. Here we demonstrated that TMEM39A, also called SUSR2, modulates autophagy activity by regulating the spatial distribution and levels of PtdIns(4)P. Depletion of SUSR2 elevates late endosomal/lysosomal PtdIns(4)P levels, facilitating recruitment of the HOPS complex to promote assembly of the SNARE complex for autophagosome maturation. SUSR2 knockdown also increases the degradative capability of lysosomes. Mechanistically, SUSR2 interacts with the ER-localized PtdIns(4)P phosphatase SAC1 and also the COPII SEC23/SEC24 subunits to promote the ER-to-Golgi transport of SAC1. Retention of SAC1 on the ER in SUSR2 knockdown cells increases the level of PtdIns(3)P produced by the VPS34 complex, promoting autophagosome formation. Our study reveals that TMEM39A/SUSR2 acts as an adaptor protein for efficient export of SAC1 from the ER and provides insights into the pathogenesis of diseases associated with TMEM39A mutations.

Apoptosis-linked gene 2 (ALG-2, also known as PDCD6) is a member of the penta-EF-hand (PEF) family of Ca2+-binding proteins. The murine gene encoding ALG-2 was originally reported to be an essential gene for apoptosis. However, the role of ALG-2 in cell death pathways has remained elusive. In the present study, we found that cell death-inducing p53 target protein 1 (CDIP1), a pro-apoptotic protein, interacts with ALG-2 in a Ca2+-dependent manner. Co-immunoprecipitation analysis of GFP-fused CDIP1 (GFP-CDIP1) revealed that GFP-CDIP1 associates with tumor susceptibility gene 101 (TSG101), a known target of ALG-2 and a subunit of endosomal sorting complex required for transport-I (ESCRT-I). ESCRT-I is a heterotetrameric complex composed of TSG101, VPS28, VPS37 and MVB12/UBAP1. Of diverse ESCRT-I species originating from four VPS37 isoforms (A, B, C, and D), CDIP1 preferentially associates with ESCRT-I containing VPS37B or VPS37C in part through the adaptor function of ALG-2. Overexpression of GFP-CDIP1 in HEK293 cells caused caspase-3/7-mediated cell death. In addition, the cell death was enhanced by co-expression of ALG-2 and ESCRT-I, indicating that ALG-2 likely promotes CDIP1-induced cell death by promoting the association between CDIP1 and ESCRT-I. We also found that CDIP1 binds to vesicle-associated membrane protein-associated protein (VAP)A and VAPB through the two phenylalanines in an acidic tract (FFAT)-like motif in the C-terminal region of CDIP1, mutations of which resulted in reduction of CDIP1-induced cell death. Therefore, our findings suggest that different expression levels of ALG-2, ESCRT-I subunits, VAPA and VAPB may have an impact on sensitivity of anticancer drugs associated with CDIP1 expression.

The switch between HIV latency and productive transcription is regulated by an auto-feedback mechanism initiated by the viral trans-activator Tat, which functions to recruit the host transcription elongation factor P-TEFb to proviral HIV. A heterodimeric complex of CDK9 and one of three cyclin T subunits, P-TEFb is expressed at vanishingly low levels in resting memory CD4+ T cells and cellular mechanisms controlling its availability are central to regulation of the emergence of HIV from latency. Using a well-characterized primary T-cell model of HIV latency alongside healthy donor memory CD4+ T cells, we characterized specific T-cell receptor (TCR) signaling pathways that regulate the generation of transcriptionally active P-TEFb, defined as the coordinate expression of cyclin T1 and phospho-Ser175 CDK9. Protein kinase C (PKC) agonists, such as ingenol and prostratin, stimulated active P-TEFb expression and reactivated latent HIV with minimal cytotoxicity, even in the absence of intracellular calcium mobilization with an ionophore. Unexpectedly, inhibition-based experiments demonstrated that PKC agonists and TCR-mobilized diacylglycerol signal through MAP kinases ERK1/2 rather than through PKC to effect the reactivation of both P-TEFb and latent HIV. Single-cell and bulk RNA-seq analyses revealed that of the four known isoforms of the Ras guanine nucleotide exchange factor RasGRP, RasGRP1 is by far the predominantly expressed diacylglycerol-dependent isoform in CD4+ T cells. RasGRP1 should therefore mediate the activation of ERK1/2 via Ras-Raf signaling upon TCR co-stimulation or PKC agonist challenge. Combined inhibition of the PI3K-mTORC2-AKT-mTORC1 pathway and the ERK1/2 activator MEK prior to TCR co-stimulation abrogated active P-TEFb expression and substantially suppressed latent HIV reactivation. Therefore, contrary to prevailing models, the coordinate reactivation of P-TEFb and latent HIV in primary T cells following either TCR co-stimulation or PKC agonist challenge is independent of PKC but rather involves two complementary signaling arms of the TCR cascade, namely, RasGRP1-Ras-Raf-MEK-ERK1/2 and PI3K-mTORC2-AKT-mTORC1.