Structural Dissection of Epsin-1 N-Terminal Helical Peptide: The Role of Hydrophobic Residues in Modulating Membrane Curvature.

Spatiotemporal structural alterations in cellular membranes are the hallmark of many vital processes. In these cellular events, the induction of local changes in membrane curvature often plays a pivotal role. Many amphiphilic peptides are able to modulate membrane curvature, but there is little information on specific structural factors that direct the curvature change. Epsin-1 is a representative protein thought to initiate invagination of the plasma membrane upon clathrin-coated vesicles formation. Its N-terminal helical segment (EpN18) plays a key role in inducing positive membrane curvature. This study aimed to elucidate the essential structural features of EpN18 in order to better understand general curvature-inducing mechanisms, and to design effective tools for rationally controlling membrane curvature. Structural dissection of peptides derived from EpN18 revealed the decisive contribution of hydrophobic residues to (i) enhancing membrane interactions, (ii) helix structuring, (iii) inducing positive membrane curvature, and (iv) loosening lipid packing. The strongest effect was obtained by substitution with leucine residues, as this EpN18 analog showed a marked ability to promote the influx of octa-arginine cell-penetrating peptides into living cells.

The clinicopathologic significance of Tks5 expression of peritoneal mesothelial cells in gastric cancer patients.

BACKGROUND: Gastric cancer (GC) patients frequently develop peritoneal metastasis. Recently, it has been reported that peritoneal mesothelial cells (PMCs) activated by GC cells acquire a migratory capacity and promote GC cell invasion. The invasiveness of PMCs reportedly depends on the activity of Tks5, an adaptor protein required for invadopodia formation. However, the relationship between clinicopathologic features and Tks5 expression in PMCs has been poorly documented. In this study, we evaluated the clinicopathologic significance of the Tks5 expression of PMCs in GC patients. MATERIALS AND METHODS: A total of 110 GC patients who underwent gastrectomy were enrolled in this study. Tks5 expressions in PMCs from the greater omentum, lesser omentum and retroperitoneum were evaluated by immunohistochemistry. We analyzed the correlation between Tks5 expressions in PMCs and the patients' clinicopathologic features. RESULTS: Tks5 expression was found in 71 (64.5%) of the 110 patients, while 39 (35.5%) were Tks5-negative. Tks5 positivity was significantly (p = 0.038) associated with a greater tumor depth (i.e., T3/4 compared with T1/T2). Peritoneal recurrence was found in 12 of 98 cases within 3 years of surgery. The 3-year peritoneal recurrence-free survival (PRFS) rate in Tks5-positive cases was significantly poorer than that in Tks5-negative cases (80.1% vs 97.4%, p = 0.024). Multivariate analysis revealed that Tks5 positivity and lymph node metastasis were independent factors for PRFS. CONCLUSION: Tks5 is frequently expressed in PMCs in advanced-stage gastric cancer. Tks5 might be a useful predictor for peritoneal recurrence in GC patients.

Key proteins of invadopodia are overexpressed in oral squamous cell carcinoma suggesting an important role of MT1-MMP in the tumoral progression.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is the most relevant malignant neoplasm among all head and neck tumours due to its high prevalence and unfavourable prognosis. Tumour invasion and metastasis that affect prognosis are result of a set of complex events that cells with invasive potential use to spread to other regions. These cells use several mechanisms to invade tissues, including a type of finger-like membrane protrusion called invadopodia. This study aims to investigate the immunoexpression of invaopodia related-proteins TKs5, cortactin, TKs4 and MT1-MMP in OSCC and correlate it to clinicopathological data. METHODS: An immunohistochemical evaluation of fifty cases of OSCCs and 20 cases of oral mucosa (OM) were assessed. The expression of invadopodia proteins were analysed in comparison to normal tissue (OM) and correlated to different clinical-stage and histological grade of OSCC. RESULTS: TKs5, cortactin, TKs4 and MT1-MMP were significantly overexpressed in OSCC when compared to OM (p < 0.0001). Among tumour stages, TKs5 showed a statistical difference in immunolabelling between stage I and III (p = 0.026). Cortactin immunolabelling was statistically higher in grade I than in grade II and III. No differences were seen on TKs4 expression based on tumour staging or grading. MT1-MMP was higher expressed and showed statistical difference between stages I and III and grades I compared to II and III. CONCLUSIONS: The invadopodia related-proteins were found to be overexpressed in OSCC when compared to OM, suggesting invadopodia formation and activity. Besides overexpressed in OSCC, cortactin, TKs4 and TKs5 showed no or ambiguous differences in protein expression when compared among clinical-stages or histological grades groups. Conversely, the expression of MT1-MMP increased in advanced stages and less differentiated tumours, suggesting MT1-MMP expression as a promising prognostic marker in OSCC.

Genetically regulated expression underlies cellular sensitivity to chemotherapy in diverse populations.

Most cancer chemotherapeutic agents are ineffective in a subset of patients; thus, it is important to consider the role of genetic variation in drug response. Lymphoblastoid cell lines (LCLs) in 1000 Genomes Project populations of diverse ancestries are a useful model for determining how genetic factors impact the variation in cytotoxicity. In our study, LCLs from three 1000 Genomes Project populations of diverse ancestries were previously treated with increasing concentrations of eight chemotherapeutic drugs, and cell growth inhibition was measured at each dose with half-maximal inhibitory concentration (IC50) or area under the dose-response curve (AUC) as our phenotype for each drug. We conducted both genome-wide association studies (GWAS) and transcriptome-wide association studies (TWAS) within and across ancestral populations. We identified four unique loci in GWAS and three genes in TWAS to be significantly associated with the chemotherapy-induced cytotoxicity within and across ancestral populations. In the etoposide TWAS, increased STARD5 predicted expression associated with decreased etoposide IC50 (P = 8.5 × 10-8). Functional studies in A549, a lung cancer cell line, revealed that knockdown of STARD5 expression resulted in the decreased sensitivity to etoposide following exposure for 72 (P = 0.033) and 96 h (P = 0.0001). By identifying loci and genes associated with cytotoxicity across ancestral populations, we strive to understand the genetic factors impacting the effectiveness of chemotherapy drugs and to contribute to the development of future cancer treatment.

Cortical Proteins Associated With Cognitive Resilience in Community-Dwelling Older Persons.

Importance: Identifying genes and proteins for cognitive resilience (ie, targets that may be associated with slowing or preventing cognitive decline regardless of the presence, number, or combination of common neuropathologic conditions) provides a complementary approach to developing novel therapeutics for the treatment and prevention of Alzheimer disease and related dementias. Objective: To identify proteins associated with cognitive resilience via a proteome-wide association study of the human dorsolateral prefrontal cortex. Design, Setting, and Participants: This study used data from 391 community-dwelling older persons who participated in the Religious Orders Study and the Rush Memory and Aging Project. The Religious Orders Study began enrollment January 1, 1994, and the Rush Memory and Aging Project began enrollment September 1, 1997, and data were collected and analyzed through October 23, 2019. Exposures: Participants had undergone annual detailed clinical examinations, postmortem evaluations, and tandem mass tag proteomics analyses. Main Outcomes and Measures: The outcome of cognitive resilience was defined as a longitudinal change in cognition over time after controlling for common age-related neuropathologic indices, including Alzheimer disease, Lewy bodies, transactive response DNA-binding protein 43, hippocampal sclerosis, infarcts, and vessel diseases. More than 8000 high abundance proteins were quantified from frozen dorsolateral prefrontal cortex tissue using tandem mass tag and liquid chromatography-mass spectrometry. Results: There were 391 participants (273 women); their mean (SD) age was 79.7 (6.7) years at baseline and 89.2 (6.5) years at death. Eight cortical proteins were identified in association with cognitive resilience: a higher level of NRN1 (estimate, 0.140; SE, 0.024; P = 7.35 × 10-9), ACTN4 (estimate, 0.321; SE, 0.065; P = 9.94 × 10-7), EPHX4 (estimate, 0.198; SE, 0.042; P = 2.13 × 10-6), RPH3A (estimate, 0.148; SE, 0.031; P = 2.58 × 10-6), SGTB (estimate, 0.211; SE, 0.045; P = 3.28 × 10-6), CPLX1 (estimate, 0.136; SE, 0.029; P = 4.06 × 10-6), and SH3GL1 (estimate, 0.179; SE, 0.039; P = 4.21 × 10-6) and a lower level of UBA1 (estimate, -0.366; SE, 0.076; P = 1.43 × 10-6) were associated with greater resilience. Conclusions and Relevance: These protein signals may represent novel targets for the maintenance of cognition in old age.

Biomarkers of vascular disease in diabetes: the adipose-immune system cross talk.

Experimental and clinical studies aimed at investigating the mechanism(s) underlying vascular complications of diabetes indicate that a great number of molecules are involved in the pathogenesis of these complications. Most of these molecules are inflammatory mediators or markers generated by immune or adipose tissue. Some of them, i.e. resistin and sortilin, have been shown to be involved in the cross talk between adipocytes and inflammatory cells. This interaction is an attractive area of research, particularly in type 2 diabetes and obesity. Other proteins, such as adiponectin and visfatin, appear to be more promising as possible vascular markers. In addition, some molecules involved in calcium/phosphorus metabolism, such as klotho and FGF23, have an involvement in the pathogenesis of diabetic vasculopathy, which appears to be dependent on the degree of vascular impairment. Inflammatory markers are a promising tool for treatment decisions while measuring plasma levels of adipokines, sortilin, Klotho and FGF23 in adequately sized longitudinal studies is expected to allow a more precise characterization of diabetic vascular disease and the optimal use of personalized treatment strategies.

PAK Kinases Target Sortilin and Modulate Its Sorting.

The multifunctional type 1 receptor sortilin is involved in endocytosis and intracellular transport of ligands. The short intracellular domain of sortilin binds several cytoplasmic adaptor proteins (e.g., the AP-1 complex and GGA1 to -3), most of which target two well-defined motifs: a C-terminal acidic cluster dileucine motif and a YXXΦ motif in the proximal third of the domain. Both motifs contribute to endocytosis as well as Golgi-endosome trafficking of sortilin. The C-terminal acidic cluster harbors a serine residue, which is subject to phosphorylation by casein kinase. Phosphorylation of this serine residue is known to modulate adaptor binding to sortilin. Here, we show that the cytoplasmic domain of sortilin also engages Rac-p21-activated kinases 1 to 3 (PAK1-3) via a binding segment that includes a tyrosine-based motif, also encompassing a serine residue. We further demonstrate that PAK1-3 specifically phosphorylate this serine residue and that this phosphorylation alters the affinity for AP-1 binding and consequently changes the intracellular localization of sortilin as a result of modulated trafficking. Our findings suggest that trafficking of ligands bound to sortilin is in part regulated by group A PAK kinases, which are downstream effectors of Rho GTPases and are known to affect a variety of processes by remodeling the cytoskeleton and by promoting gene transcription and cell survival.

Binding of thyroglobulin (Tg) to the low-density lipoprotein receptor-associated protein (RAP) during the biosynthetic pathway prevents premature Tg interactions with sortilin.

OBJECTIVE: Sortilin, a Vps10p family member, is expressed by thyroid epithelial cells (TEC), where it binds to internalized thyroglobulin (Tg) molecules. Premature binding of Tg to sortilin during biosynthesis may cause intracellular retention of Tg. Such a premature interaction may be prevented by one or more inhibitor/s. Because both sortilin and Tg bind to the low-density lipoprotein receptor-associated protein (RAP), we investigated whether RAP serves such a function. METHODS: Immunofluorescence staining for sortilin, Tg, and RAP was performed in FRTL-5 cells. Co-immunoprecipitation experiments were performed in extracts from FRTL-5 or COS-7 cells, the former co-transfected with Tg and/or RAP and/or sortilin, or in thyroid extracts from RAP KO mice. RESULTS: Tg and sortilin did not co-localize in FRTL-5 cells following inhibition of protein synthesis, suggesting that newly synthesized, endogenous sortilin and Tg do not interact, in confirmation of which an anti-sortilin antibody did not co-precipitate Tg in FRTL-5 cells. In contrast, Tg co-localized with RAP in FRTL-5 cells. Co-immunoprecipitation of Tg with an anti-sortilin antibody in COS-7 cells transfected with sortilin and Tg was abolished when cells were co-transfected with RAP, indicating that RAP prevents binding of Tg to sortilin during biosynthesis, in confirmation of which an anti-sortilin antibody co-precipitated Tg in thyroid extracts from RAP KO mice to a greater extent than in thyroid extracts from WT mice. CONCLUSIONS: Tg does not bind prematurely to sortilin because of its interaction with RAP during protein biosynthesis. These findings add new information to the knowledge of thyroid physiology.

Elevated expression of STIM1 is involved in lung tumorigenesis.

This study aimed to address the potential role of STIM1 (stromal interaction molecule 1) in lung tumorigenesis. Colony formation in soft agar assay and tumorigenicity in nude mice assay were conducted. Western blot, immunohistochemistry and quantitative real-time polymerase chain reaction were used to measure the STIM1 expression. The distribution of cell cycle was detected by flow cytometry assay. Our results showed that the expression of STIM1 mRNA was significantly higher in human lung tumors than that in adjacent non-neoplastic lung tissues. Significantly increased expression of STIM1 mRNA and protein was observed in 16HBE-benzo(a)pyrene (BaP) cells and in BaP-treated mice lung tissues compared with 16HBE-control cells and the control group, respectively. Silencing STIM1 inhibited the proliferation and colony formation of A549 cells in in vitro experiments, attenuated the growth of tumor xenografts of A549 cells in in vivo experiments and induced the arrest of cell cycle in the G1 phase. The markedly decreased expression of cyclin D1 protein was observed in A549-shRNA-STIM1 cells as compared to A549-shRNA-control cells. The markedly increased expression of p21 protein was observed in A549-shRNA-STIM1 cells as compared to A549-shRNA-control cells. The expression levels of ß-catenin and TGIF proteins were lower in A549-shRNA-STIM1 cells than those in A549-shRNA-control cells. In conclusion, this study indicated that the elevated expression of STIM1 might be involved in lung tumorigenesis.

Production and Characterization of a Novel Monoclonal Antibody Against Human Sortilin.

Sortilin, as a member of Vps10p-domain sorting receptor family, is overexpressed in a number of malignancies, including ovarian carcinoma. Antibodies against sortilin may contribute to further clarification of sortilin functional activities in signal transduction, intracellular sorting of proteins, and endocytosis. The aim of this study was to produce a monoclonal antibody against a synthetic peptide derived from extracellular N-terminal region of sortilin to be used as a tool for investigating sortilin characteristics in ovarian carcinoma. A synthetic peptide derived from the last 50 amino acids of extracellular domain of sortilin protein was selected and conjugated to keyhole limpet hemocyanin and used to immunize mice. The anti-sortilin monoclonal antibody (MAb), clone 2D8, was purified from supernatant of final hybridoma clone using peptide-affinity chromatography column. Reactivity of antibody with the immunizing peptide was assessed in ELISA. Furthermore, flow cytometry and Western blot analyses were used to investigate the reactivity of antibody with its target in a panel of ovarian carcinoma cell lines or tissues. MAb 2D8 was able to recognize the coated immunizing peptide in ELISA and detect its protein target, sortilin, in flow cytometry and Western blot analyses. The achieved data suggest that the developed monoclonal antibody may be applicable as a research tool for detection of sortilin protein in Western blot as well as flow cytometry tests.

Roles of bovine viral diarrhea virus envelope glycoproteins in inducing autophagy in MDBK cells.

Macroautophagy (autophagy) is an evolutionarily conserved control process that maintains cellular homeostasis in eukaryotic cells. Autophagy principally serves an adaptive role to degrade dysfunctional proteins and to clean damaged organelles in response to pathogenic, viral, or microbial infection, nutrient deprivation and endoplasmic reticulum (ER) stress. In previous study, we showed bovine viral diarrhea virus (BVDV) NADL infection induced autophagy and significantly elevated the expression levels of autophagy-related genes, Beclin1 and ATG14, at 12 h post-infection in MDBK cells. However, the specific mechanisms involved in controlling autophagic activity remain unclear. Here, we investigate the effects of BVDV NADL envelope glycoproteins overexpression on inducing autophagy. The results show that viral envelope glycoproteins E(rns) and E2 overexpression mediated by lentivirus increase the formation of autophagosome, the percentage of GFP-LC3 puncta-positive cells and the expression levels of Beclin1 and ATG14. Whereas E1 overexpression doesn't affect autophagic activity. Collectively, these findings suggest that the viral envelope glycoproteins E(rns) and E2 are involved in inducing autophagy, and provide a mechanistic insight into the regulation of autophagy in viral infected cells.

Fractionation profiling: a fast and versatile approach for mapping vesicle proteomes and protein-protein interactions.

We developed "fractionation profiling," a method for rapid proteomic analysis of membrane vesicles and protein particles. The approach combines quantitative proteomics with subcellular fractionation to generate signature protein abundance distribution profiles. Functionally associated groups of proteins are revealed through cluster analysis. To validate the method, we first profiled >3500 proteins from HeLa cells and identified known clathrin-coated vesicle proteins with >90% accuracy. We then profiled >2400 proteins from Drosophila S2 cells, and we report the first comprehensive insect clathrin-coated vesicle proteome. Of importance, the cluster analysis extends to all profiled proteins and thus identifies a diverse range of known and novel cytosolic and membrane-associated protein complexes. We show that it also allows the detailed compositional characterization of complexes, including the delineation of subcomplexes and subunit stoichiometry. Our predictions are presented in an interactive database. Fractionation profiling is a universal method for defining the clathrin-coated vesicle proteome and may be adapted for the analysis of other types of vesicles and particles. In addition, it provides a versatile tool for the rapid generation of large-scale protein interaction maps.

Sorting receptor sortilin-a culprit in cardiovascular and neurological diseases.

Sortilin is a sorting receptor that directs target proteins, such as growth factors, signaling receptors, and enzymes, to their destined location in secretory or endocytic compartments of cells. The activity of sortilin is essential for proper function of not only neurons but also non-neuronal cell types, and receptor (dys)function emerges as a major cause of malignancies, including hypercholesterolemia, retinal degeneration, neuronal cell loss in stroke and spinal cord injury, or Alzheimer's disease and other neurodegenerative disorders. In this article, we describe the molecular mechanisms of sortilin action in protein sorting and signaling and how modulation of receptor function may offer novel therapeutic strategies for treatment of common diseases of the cardiovascular and nervous systems.

Linking transcriptional changes over time in stimulated dendritic cells to identify gene networks activated during the innate immune response.

The innate immune response is primarily mediated by the Toll-like receptors functioning through the MyD88-dependent and TRIF-dependent pathways. Despite being widely studied, it is not yet completely understood and systems-level analyses have been lacking. In this study, we identified a high-probability network of genes activated during the innate immune response using a novel approach to analyze time-course gene expression profiles of activated immune cells in combination with a large gene regulatory and protein-protein interaction network. We classified the immune response into three consecutive time-dependent stages and identified the most probable paths between genes showing a significant change in expression at each stage. The resultant network contained several novel and known regulators of the innate immune response, many of which did not show any observable change in expression at the sampled time points. The response network shows the dominance of genes from specific functional classes during different stages of the immune response. It also suggests a role for the protein phosphatase 2a catalytic subunit α in the regulation of the immunoproteasome during the late phase of the response. In order to clarify the differences between the MyD88-dependent and TRIF-dependent pathways in the innate immune response, time-course gene expression profiles from MyD88-knockout and TRIF-knockout dendritic cells were analyzed. Their response networks suggest the dominance of the MyD88-dependent pathway in the innate immune response, and an association of the circadian regulators and immunoproteasomal degradation with the TRIF-dependent pathway. The response network presented here provides the most probable associations between genes expressed in the early and the late phases of the innate immune response, while taking into account the intermediate regulators. We propose that the method described here can also be used in the identification of time-dependent gene sub-networks in other biological systems.

Prognostic significance of Tks5 expression in gliomas.

A pathological hallmark of gliomas is their extensive invasion into the brain parenchyma regardless of tumour grade. Clinically this is a major factor in tumour recurrence as surgery and adjuvant therapies are unable to eradicate all the infiltrating malignant cells. Tyrosine kinase substrate with five SH3 domains (Tks5, also known as SH3PXD2A) and cortactin are required for the formation of invadopodia, actin-based protrusions of tumour cells with associated proteolytic activity implicated in tumour invasion. We investigated the prognostic significance of Tks5 and cortactin expression in 57 patients with various grades of glioma. Expression of Tks5 or cortactin occurred in all grades of tumours and expression of Tks5, but not cortactin, was associated with significantly reduced patient survival among glioma patients. This association was clearest in patients with low-grade astrocytomas and oligoastrocytomas. These results suggest a prognostic relevance for the Tks5 invadopodial protein in glial-derived brain tumours.

Interactions among the SNARE proteins and complexin analyzed by a yeast four-hybrid assay.

Exocytosis is one of the most crucial and ubiquitous processes in all of biology. This event is mediated by the formation of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complexes, ternary assemblies of syntaxin, SNAP23/SNAP25 (synaptosomal-associated protein of 23 or 25 kDa), and synaptobrevin. The exocytotic process can be further regulated by complexin, which interacts with the SNARE complex. Complexin is involved in a Ca(2+)-triggered exocytotic process. In eukaryotic cells, multiple isoforms of SNARE proteins are expressed and are involved in distinct types of exocytosis. To understand the underlying biochemical mechanism of various exocytotic processes mediated by different SNARE protein isoforms, we systematically analyzed the interactions among syntaxin, SNAP23/SNAP25, synaptobrevin, and complexin by employing a newly developed yeast four-hybrid interaction assay. The efficiency of SNARE complex formation and the specificity of complexin binding are regulated by the different SNARE protein isoforms. Therefore, various types of exocytosis, occurring on different time scales with different efficiencies, can be explained by the involved SNARE complexes composed of different combinations of SNARE protein isoforms.

Conserved oligomeric Golgi complex specifically regulates the maintenance of Golgi glycosylation machinery.

Cell surface lectin staining, examination of Golgi glycosyltransferases stability and localization, and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis were employed to investigate conserved oligomeric Golgi (COG)-dependent glycosylation defects in HeLa cells. Both Griffonia simplicifolia lectin-II and Galanthus nivalus lectins were specifically bound to the plasma membrane glycoconjugates of COG-depleted cells, indicating defects in activity of medial- and trans-Golgi-localized enzymes. In response to siRNA-induced depletion of COG complex subunits, several key components of Golgi glycosylation machinery, including MAN2A1, MGAT1, B4GALT1 and ST6GAL1, were severely mislocalized. MALDI-TOF analysis of total N-linked glycoconjugates indicated a decrease in the relative amount of sialylated glycans in both COG3 KD and COG4 KD cells. In agreement to a proposed role of the COG complex in retrograde membrane trafficking, all types of COG-depleted HeLa cells were deficient in the Brefeldin A- and Sar1 DN-induced redistribution of Golgi resident glycosyltransferases to the endoplasmic reticulum. The retrograde trafficking of medial- and trans-Golgi-localized glycosylation enzymes was affected to a larger extent, strongly indicating that the COG complex regulates the intra-Golgi protein movement. COG complex-deficient cells were not defective in Golgi re-assembly after the Brefeldin A washout, confirming specificity in the retrograde trafficking block. The lobe B COG subcomplex subunits COG6 and COG8 were localized on trafficking intermediates that carry Golgi glycosyltransferases, indicating that the COG complex is directly involved in trafficking and maintenance of Golgi glycosylation machinery.

The absence of a clathrin adapter confers unique polarity essential to proximal tubule function.

It is well established that many cognate basolateral plasma membrane proteins are expressed apically in proximal tubule cells thus optimizing the reabsorption capacity of the kidney. The protein clathrin and its adapter proteins normally regulate basolateral polarity. Here we tested whether the unique proximal tubule polarity is dependent on an epithelial-specific basolateral clathrin adapter, AP1B, present in most other epithelia. Quantitative PCR of isolated mouse renal tubules showed that AP1B was absent in proximal tubules but present in medullary and cortical thick ascending limbs of Henle, and cortical collecting ducts. Western blot confirmed the absence of AP1B in three established proximal tubule cell lines. Knockdown of AP1B by shRNA in prototypical distal tubule MDCK cells resulted in redistribution of the basolateral parathyroid hormone receptor, the insulin-like growth factor II receptor/calcium-independent mannose-6-phosphate receptor, and the junctional adhesion molecule, JAM-C, to a proximal tubule-like nonpolar localization. Yeast two-hybrid assays detected direct interactions between the cytoplasmic tails of these plasma membrane proteins and the cargo-binding region of the AP1B complex. Hence, our results show that differential expression of AP1B contributes to normal kidney function and illustrates possible roles of this adapter protein in kidney development, physiology, and pathology.

AP180-mediated trafficking of Vamp7B limits homotypic fusion of Dictyostelium contractile vacuoles.

Clathrin-coated vesicles play an established role in endocytosis from the plasma membrane, but they are also found on internal organelles. We examined the composition of clathrin-coated vesicles on an internal organelle responsible for osmoregulation, the Dictyostelium discoideum contractile vacuole. Clathrin puncta on contractile vacuoles contained multiple accessory proteins typical of plasma membrane-coated pits, including AP2, AP180, and epsin, but not Hip1r. To examine how these clathrin accessory proteins influenced the contractile vacuole, we generated cell lines that carried single and double gene knockouts in the same genetic background. Single or double mutants that lacked AP180 or AP2 exhibited abnormally large contractile vacuoles. The enlarged contractile vacuoles in AP180-null mutants formed because of excessive homotypic fusion among contractile vacuoles. The SNARE protein Vamp7B was mislocalized and enriched on the contractile vacuoles of AP180-null mutants. In vitro assays revealed that AP180 interacted with the cytoplasmic domain of Vamp7B. We propose that AP180 directs Vamp7B into clathrin-coated vesicles on contractile vacuoles, creating an efficient mechanism for regulating the internal distribution of fusion-competent SNARE proteins and limiting homotypic fusions among contractile vacuoles. Dictyostelium contractile vacuoles offer a valuable system to study clathrin-coated vesicles on internal organelles within eukaryotic cells.