Carboxypeptidase Y Research Articles

Inhibition of Lysosomal Cathepsin A and Neuraminidase 1 Interaction by Anti-Obesity Cyclic Peptide.

Chronic inflammation in adipose tissue is associated with metabolic disorders such as obesity and type 2 diabetes. Novel small molecules targeting adipocyte differentiation and fat accumulation offer potential for new anti-inflammatory and anti-obesity drugs. Here we show that the marine cyclic heptapeptide stylissatin A and its analogs (SAs) inhibit membranous neuraminidase 1 (Neu1) function by interacting with lysosomal protective protein cathepsin A (PPCA). Neu1 has been less explored as a therapeutic target due to the genetic defects leading to neurodegenerative disorders. However, unlike traditional neuraminidase inhibitors, SAs don't directly bind to Neu1 but modulate the molecular chaperone activity of PPCA. SAs caused degradation of perilipin 1 around lipid droplets and inhibited fat accumulation, along with decrease in membranous Neu1. Molecular docking and molecular dynamics simulations revealed that SAs interacted with activated PPCA at the Neu1 binding site. Focusing on this newfound protein-protein interaction inhibition mechanism could lead to the development of pharmaceuticals with fewer side effects.

A DUF3844 domain-containing protein is required for vacuolar protein sorting in Schizosaccharomyces pombe.

Protein trafficking to vacuoles in plants and fungi, and to lysosomes in animals, is essential for the maintenance of cellular homeostasis. In Saccharomyces cerevisiae, the vacuolar protein sorting (VPS) pathway has been well studied by using vacuolar carboxypeptidase Y (CPY) as a model, and many VPS genes have been identified. By contrast, the vacuolar protein trafficking pathway in Schizosaccharomyces pombe remains poorly understood. In this study, we identified a novel VPS gene (SPBC1709.03) in S. pombe that is broadly conserved in fungi, but not in S. cerevisiae. Owing to its DUF3844 domain of unknown function, the gene was named vps3844. Disruption mutants of vps3844 had defects in both CPY sorting and incorporation of FM4-64 dye into the vacuolar membrane. Partial deletion analysis of the Vps3844 protein revealed that, within the DUF3844 domain, the region comprising amino acids 354 to 380 is important for protein trafficking to the vacuole. Our findings represent the first report of a VPS gene involved in vacuolar transport that is conserved in fungi, particularly S. pombe, but lacks representation in S. cerevisiae.

3-[5-(1H-Indol-3-ylmethylene)-4-oxo-2-thioxothiazolidin-3-yl]-propionic Acid as a Potential Polypharmacological Agent

Searching for new types of biological activities among preliminarily identified hit compounds is a key challenge in modern medicinal chemistry. In our study, a previously studied 3-[5-(1H-indol-3-ylmethylene)-4-oxo-2-thioxothiazolidin-3-yl]-propionic acid (Les-6614) was screened for antimicrobial, antifungal, anti-allergic, and antitumor activities. Moreover, cytotoxicity, molecular docking, and SwissAdme online target screening were accomplished. It was determined that the Les-6614 has slight antimicrobial and antitumor activity. However, the studied compound decreased IgE levels in sensitized guinea pigs by 33–86% and reduced IgA, IgM, IL-2, and TNF-α, indicating anti-inflammatory and anti-allergic aactivities. According to the SwissADME web tool, target predictions for Les-6614 potentially have an affinity for lysosomal protective protein, Thromboxane-A synthase, and PPARγ. The molecular docking confirmed that the studied 2-thioxo-4-thiazolidinone derivative showed good bonding with LLP and TXAS, leading to stable protein–ligand complexes. Additionally, Les-6614 is a potential PPARγ modulator, which is important in the pathogenesis of allergy, cancer, and cardiovascular diseases.

Copper inhibits protein maturation in the secretory pathway by targeting the Sec61 translocon in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, proteins destined for secretion utilize the post-translational translocon machinery to gain entry into the endoplasmic reticulum. These proteins then mature by undergoing a number of post-translational modifications in different compartments of the secretory pathway. While these modifications have been well established for many proteins, to date only a few studies have been conducted regarding the conditions and factors affecting maturation of these proteins before entering into the endoplasmic reticulum. Here, using immunoblotting, microscopy, and spot test assays, we show that excess copper inhibits the Sec61 translocon function and causes accumulation of two well-known post-translationally translocated proteins, Gas1 (glycophospholipid-anchored surface protein) and CPY (carboxypeptidase Y), in the cytosol. We further show that the copper-sensitive phenotype of sec61-deficient yeast cells is ameliorated by restoring the levels of SEC61 through plasmid transformation. Furthermore, screening of translocation-defective Sec61 mutants revealed that sec61-22, bearing L80M, V134I, M248V, and L342S mutations, is resistant to copper, suggesting that copper might be inflicting toxicity through one of these residues. In conclusion, these findings imply that copper-mediated accumulation of post-translationally translocated proteins is due to the inhibition of Sec61.

Rapid inactivation of the yeast Sec complex selectively blocks transport of post-translationally translocated proteins

The yeast endoplasmic reticulum has three distinct protein translocation channels. The heterotrimeric Sec61 and Ssh1 complexes, which bind translating ribosomes, mediate cotranslational translocation of proteins targeted to the endoplasmic reticulum by the signal recognition particle (SRP) and SRP receptor targeting pathway, whereas the heptameric Sec complex has been proposed to mediate ribosome-independent post-translational translocation of proteins with less hydrophobic signal sequences that escape recognition by the SRP. However, multiple reports have proposed that the Sec complex may function cotranslationally and be involved in translocation or integration of SRP-dependent protein translocation substrates. To provide insight into these conflicting views, we induced expression of the tobacco etch virus protease to achieve rapid inactivation of the Sec complex by protease-mediated cleavage within the cytoplasmic domain of the Sec63 protein. Protein translocation assays conducted after tobacco etch virus protease induction revealed a complete block in translocation of two well-characterized substrates of the Sec complex, carboxypeptidase Y (CPY) and Gas1p, when the protease cleavage sites were located at structural domain boundaries in Sec63. However, integration of SRP-dependent membrane protein substrates was not detectably impacted. Moreover, redirecting CPY to the cotranslational pathway by increasing the hydrophobicity of the signal sequence rendered translocation of CPY insensitive to inactivation of the Sec complex. We conclude that the Sec complex is primarily responsible for the translocation of yeast secretome proteins with marginally hydrophobic signal sequences.

Spc1 regulates the signal peptidase-mediated processing of membrane proteins.

ABSTRACTSignal peptidase (SPase) cleaves the signal sequences (SSs) of secretory precursors. It contains an evolutionarily conserved membrane protein subunit, Spc1, that is dispensable for the catalytic activity of SPase and whose role remains unknown. In this study, we investigated the function of yeast Spc1. First, we set up an in vivo SPase cleavage assay using variants of the secretory protein carboxypeptidase Y (CPY) with SSs modified in the N-terminal and hydrophobic core regions. When comparing the SS cleavage efficiencies of these variants in cells with or without Spc1, we found that signal-anchored sequences became more susceptible to cleavage by SPase without Spc1. Furthermore, SPase-mediated processing of model membrane proteins was enhanced in the absence of Spc1 and was reduced upon overexpression of Spc1. Spc1 co-immunoprecipitated with proteins carrying uncleaved signal-anchored or transmembrane (TM) segments. Taken together, these results suggest that Spc1 protects TM segments from SPase action, thereby sharpening SPase substrate selection and acting as a negative regulator of the SPase-mediated processing of membrane proteins.

Intracellular protein delivery using QRPL – A vacuolar targeting signal on carboxypeptidase Y

The signal peptide sequence is known to increase transport efficiency to organelles in eukaryotic cells. In this study, we focus on the signal peptide of the vacuolar protein for vacuolar targeting. The signal peptide sequence QRPL of carboxypeptidase Y (CPY) was inserted inside the interest protein that does not locate in the vacuole for vacuolar targeting. We constructed recombinant strains MBTL-Q-DJ1 and MBTL-Q-DJ2 containing QRPL and green florescent protein (GFP) or aldehyde dehydrogenase 6 (ALD6), respectively. The protein location was then confirmed by confocal microscopy. Fascinatingly, the green fluorescent protein that contains QRPL inside the sequence could be expressed faster than its natural form (within 1 h after induction). Also, the aldehyde removal activity of ALD6 protein in the recombinant yeast was then analyzed by measuring the luminescent intensity in Vibrio fischeri. We confirmed that MBTL-Q-DJ2 containing ALD6 protein has the aldehydes-reducing ability, and in particular, the highest efficiency showed at 500 μg/μL of vacuolar enzyme. In summary, the signal peptide QRPL could be used not only to transport proteins accurately to vacuole but also to improve the protein activity and shorten the induction time.

Chimeric Peptides Self-Assembling on Titanium Carbide MXenes as Biosensing Interfaces for Activity Assay of Post-translational Modification Enzymes.

Post-translational modifications (PTMs) refer to the chemical modifications of proteins coordinated by PTM enzymes, and they play a key role in numerous physiological and pathological processes. Herein, chimeric peptide-functionalized titanium carbide MXenes (Pep-Ti3C2) were devised for the activity assay of PTM enzymes by integration with carboxypeptidase Y (CPY)-mediated peptide cleavage. The Pep-Ti3C2 is fabricated by self-assembly of chimeric peptide probes on the surface of phospholipid-coated Ti3C2 MXenes and works as the fluorescent nanoprobe for the sensing of PTM enzymes. In the presence of a target PTM enzyme, the modification groups in the peptide probes are removed along with the digestion of the peptides by CPY, thereby leading to the release of labeled fluorophores. Consequently, fluorescent analysis of PTM enzymes, including deacetylase sirtuin-1 and protein phosphatase 2C at low-nanomolar concentrations was achieved. Furthermore, the versatility of the nanoprobes was also demonstrated in simultaneous profiling of the activities of the two PTM enzymes in different cells, as well as in evaluation of the inhibition on PTMs by small molecules in complicated biological samples. Therefore, this work deploys peptide-functionalized MXenes as a generic biosensing interface for the activity assay of PTM enzymes, providing a useful tool for biochemical research and clinical diagnosis.

Crosstalk between protein N-glycosylation and lipid metabolism in Saccharomyces cerevisiae

The endoplasmic reticulum (ER) is a multi functional organelle and plays a crucial role in protein folding and lipid biosynthesis. The SEC59 gene encodes dolichol kinase, required for protein glycosylation in the ER. The mutation of sec59-1 caused a protein N-glycosylation defect mediated ER stress resulting in increased levels of phospholipid, neutral lipid and sterol, whereas growth was reduced. In the sec59-1∆ cell, the N-glycosylation of vacuolar carboxy peptidase-Y (CPY) was significantly reduced; whereas the ER stress marker Kar2p and unfolded protein response (UPR) were significantly increased. Increased levels of Triacylglycerol (TAG), sterol ester (SE), and lipid droplets (LD) could be attributed to up-regulation of DPP1, LRO1, and ARE2 in the sec 59-1∆ cell. Also, the diacylglycerol (DAG), sterol (STE), and free fatty acids (FFA) levels were significantly increased, whereas the genes involved in peroxisome biogenesis and Pex3-EGFP levels were reduced when compared to the wild-type. The microarray data also revealed increased expression of genes involved in phospholipid, TAG, fatty acid, sterol synthesis, and phospholipid transport resulting in dysregulation of lipid homeostasis in the sec59-1∆ cell. We conclude that SEC59 dependent N-glycosylation is required for lipid homeostasis, peroxisome biogenesis, and ER protein quality control.

Ent3 and GGA adaptors facilitate diverse anterograde and retrograde trafficking events to and from the prevacuolar endosome

Carboxypeptidases Y (Cpy1) and S (Cps1), the receptor Vps10, and the ATPase subunit Vph1 follow the carboxypeptidase Y (CPY) pathway from the trans-Golgi network (TGN) to the prevacuolar endosome (PVE). Using Schizosaccharomyces pombe quantitative live-cell imaging, biochemical and genetic analyses, we extended the previous knowledge and showed that collaboration between Gga22, the dominant Golgi-localized Gamma-ear-containing ARF-binding (GGA) protein, and Gga21, and between Gga22 and the endosomal epsin Ent3, was required for efficient: i) Vps10 anterograde trafficking from the TGN to the PVE; ii) Vps10 retrograde trafficking from the PVE to the TGN; iii) Cps1 exit from the TGN, and its sorting in the PVE en route to the vacuole; and iv) Syb1/Snc1 recycling to the plasma membrane through the PVE. Therefore, monomeric clathrin adaptors facilitated the trafficking of Vps10 in both directions of the CPY pathway, and facilitated trafficking events of Cps1 in different organelles. By contrast, they were dispensable for Vph1 trafficking. Thus, these adaptors regulated the traffic of some, but not all, of the cargo of the CPY pathway, and regulated the traffic of cargoes that do not follow this pathway. Additionally, this collaboration was required for PVE organization and efficient growth under stress.

Vac8 Controls Vacuolar Membrane Dynamics during Different Autophagy Pathways in Saccharomyces cerevisiae.

The yeast vacuole is a vital organelle, which is required for the degradation of aberrant intracellular or extracellular substrates and the recycling of the resulting nutrients as newly available building blocks for the cellular metabolism. Like the plant vacuole or the mammalian lysosome, the yeast vacuole is the destination of biosynthetic trafficking pathways that transport the vacuolar enzymes required for its functions. Moreover, substrates destined for degradation, like extracellular endocytosed cargoes that are transported by endosomes/multivesicular bodies as well as intracellular substrates that are transported via different forms of autophagosomes, have the vacuole as destination. We found that non-selective bulk autophagy of cytosolic proteins as well as the selective autophagic degradation of peroxisomes (pexophagy) and ribosomes (ribophagy) was dependent on the armadillo repeat protein Vac8 in Saccharomyces cerevisiae. Moreover, we showed that pexophagy and ribophagy depended on the palmitoylation of Vac8. In contrast, we described that Vac8 was not involved in the acidification of the vacuole nor in the targeting and maturation of certain biosynthetic cargoes, like the aspartyl-protease Pep4 (PrA) and the carboxy-peptidase Y (CPY), indicating a role of Vac8 in the uptake of selected cargoes. In addition, we found that the hallmark phenotype of the vac8Δ strain, namely the characteristic appearance of fragmented and clustered vacuoles, depended on the growth conditions. This fusion defect observed in standard glucose medium can be complemented by the replacement with oleic acid or glycerol medium. This complementation of vacuolar morphology also partially restores the degradation of peroxisomes. In summary, we found that Vac8 controlled vacuolar morphology and activity in a context- and cargo-dependent manner.

Carboxypeptidase Y activity and maintenance is modulated by a large helical structure.

Yeast carboxypeptidase Y (CPY) is a serine protease with broad substrate specificity. Structurally, CPY belongs to the α/β hydrolase fold family and contains characteristic large helices, termed the V‐shape helix, above the active site cavity. Four intramolecular disulfide bonds are located in and around the V‐shape helix. In this study, mutant CPYs were constructed in which one of these disulfide bonds was disrupted. Mutants lacking the C193–C207 bond located at the beginning of the V‐shape helix aggregated easily, while mutants lacking the C262–C268 bond located at the end of the V‐shape helix displayed decreased hydrolytic activity. The results indicate that the V‐shape helix is involved in CPY catalysis and in maintenance of its conformation.

Detection of casein kinase II by aggregation-induced emission

A novel aggregation-induced emission (AIE) probe comprised of a hydrophilic protein kinase specific peptide and a hydrophobic tetraphenylethene (TPE) unit was synthesized through click reaction. The prepared TPE-peptide probe could be completely degraded by carboxypeptidase Y (CPY) to release hydrophobic TPE part, which aggregated in buffer solution and showed strong TPE emission. In the presence of casein kinase (CKII), the phosphorylation of peptide prevented the complete degradation by CPY producing the nonemissive probe. Thus, the developed probe can be used to detect CKII homogeneously and conveniently. This detection process can be finished within 1.5 h with high sensitivity (0.05 mU/μL) and good selectivity. The developed method can also be used to screen protein kinase inhibitor even in a complex biological system.

Solid-Phase Enzymatic Peptide Synthesis to Produce an Antioxidant Dipeptide

Peptide bond synthesis is favorable to the production of bioactive small peptides. However, the abuse of toxic reagents remains an issue for chemical synthesis method, whereas the low product yield and purity limit the widespread use of enzymatic method. In this study, a new solid-phase enzymatic peptide synthesis (SPEPS) strategy was developed to produce an antioxidant tyrosine-alanine dipeptide (Tyr-Ala) by using recombinant carboxypeptidase Y (CPY) as the catalyst. The general SPEPS procedure involves three steps. First, the N-protected acyl donor was covalently attached to solid resin. Second, the peptide bond was condensed between the acyl donor and the nucleophile under the catalysis of CPY. Finally, one-step cleavage was performed to remove the protecting group and cleave the peptides from solid resin. Upon the optimization of reaction conditions, 77.92% (± 2.723%) yield of Tyr-Ala with high product purity of 90.971% (± 2.695%) was obtained. In addition, the antioxidant activity of Tyr-Ala was determined by ABTS method, indicating that the synthesized Tyr-Ala obtained by SPEPS showed a superior antioxidant capability compared with commercial glutathione.

Kinetically Controlled Carboxypeptidase-Catalyzed Synthesis of Novel Antioxidant Dipeptide Precursor BOC-Tyr-Ala

Recently, enzymatic peptide synthesis has drawn increasing attention due to its eco-friendly reagents and mild conditions, as compared to traditional chemical peptide synthesis. In this study, we successfully produced an important antioxidant dipeptide precursor, BOC-Tyr-Ala, via a kinetically controlled enzymatic peptide synthesis reaction, catalyzed by the recombinant carboxypeptidase Y (CPY) expressed in P. pastoris GS115. In this reaction, the enzyme activity was 95.043 U/mL, and we used t-butyloxycarbonyl-l-tyrosine-methyl ester (BOC-Tyr-OMe) as the acyl donor and l-alanine (l-Ala) was the amino donor. We optimized the reaction conditions to be: 30 °C, pH 9.5, organic phase (methanol)/aqueous phase = 1:20, BOC-Tyr-OMe 0.05 mol/L, Ala 0.5 mol/L, and a reaction time of 12 h. Under these conditions, the dipeptide yield reached 49.84%. Then, we established the kinetic model of the synthesis reaction in the form of Michaelis–Menten equation according to the concentration–time curve during the process and the transpeptidation mechanism. We calculated the apparent Michaelis constant $$K_{\text{m}}^{\text{app}}$$ and the apparent maximum reaction rate $$r_{\hbox{max} }^{\text{app}}$$ to be $$2.9946 \times 10^{{^{ - 2} }} {\text{ mol/L}}$$ and $$2.0406 \times 10^{ - 2} {\text{ mmol/(mL}}\,{\text{h)}}$$ , respectively.

Fluorogenic and chromogenic detection of carboxypeptidase Y with a nonpeptide-based small-molecule probe

Carboxypeptidase Y (CPY) is a vital enzyme with many well-known biological significances, such as determination of amino acid sequences and biosynthesis of polypeptides. Herein we developed a nonpeptide-based fluorescent probe to sense CPY activity accurately and successfully applied it to the inhibitory study. Based on the organic synthesis and comprehensive characterization, the refined probe CPY-P3 displayed excellent specificity towards CPY and exhibited over 400-fold enhancement of fluorescence intensity upon the hydrolysis of CPY. Compared with the commonly used peptide-based fluorescent substrate (Suc-IIW-AMC), CPY-P3 showed about 12-fold higher binding affinity and comparable catalytical efficiency for CPY. Moreover, CPY-P3 was successfully applied in CPY inhibitor characterization. To our knowledge, CPY-P3 is the first nonpeptide-based small-molecule fluorescent probe for the time-course CPY activity detection. Meanwhile, the generation of kelly color makes the detection directly visible without any complicated device. In all, this small-molecule probe should be a potentially useful tool in understanding the roles of CPY in protein sequencing and engineering, as well as the drug discovery of CPY-related diseases.

Label-Free Homogeneous Electrochemical Sensing Platform for Protein Kinase Assay Based on Carboxypeptidase Y-Assisted Peptide Cleavage and Vertically Ordered Mesoporous Silica Films.

Presented herein is a simple, robust, and label-free homogeneous electrochemical sensing platform constructed for the detection of protein kinase activity and inhibition by integration of carboxypeptidase Y (CPY)-assisted peptide cleavage reaction and vertically ordered mesoporous silica films (MSFs). In this sensing platform, the substrate peptide composed of kinase-specific recognized sequence and multiple positively charged arginine (R) residues was ingeniously designed. In the presence of protein kinase, the substrate peptide was phosphorylated and then immediately resisted CPY cleavage. The phosphorylated peptide could be effectively adsorbed on the negatively charged surface of MSFs modified indium-tin oxide (ITO) electrode (MSFs/ITO) by noncovalent electrostatic attraction. The adsorbed peptide was subsequently used as a hamper to prevent the diffusion of electroactive probe (FcMeOH) to the electrode surface through the vertically aligned nanopores, resulting in a detectable reduction of electrochemical signal. As demonstrated for the feasibility and universality of the sensing platform, both protein kinase A (PKA) and casein kinase II (CK2) were selected as the models, and the detection limits were determined to be 0.083 and 0.095 UmL-1, respectively. This sensing platform had the merits of simplicity, easy manipulation, and improved phosphorylation and cleavage efficiency, which benefited from homogeneous solution reactions without sophisticated modification or immobilization procedures. In addition, given the key role of inhibition and protein kinase activity detection in cell lysates, this proposed sensing platform showed great potential in kinase-related bioanalysis and clinical biomedicine.

The first report of cathepsin A gene in a teleost (rock bream, Oplegnathus fasciatus): An investigation of immune functions upon infection with several pathogens

We isolated and characterised a cDNA encoding the lysosomal protective protein (serine protease) cathepsin A (CTSA) from rock bream (Oplegnathus fasciatus). The full-length rock bream CTSA (RbCTSA) cDNA (1814 bp) contains an open reading frame of 1419 bp that encodes 472 amino acids. Alignment of multiple CTSA protein sequences revealed that the active site serine and histidine residues were well-conserved among the other CTSA sequences. RbCTSA is highly expressed in the peripheral blood leukocytes, kidney, spleen, liver, intestine, gill, heart, brain, stomach, and eye. RbCTSA expression was also examined in several tissues, including whole kidneys and spleens, under bacterial and viral challenge. In general, all of the examined tissues that were infected with Edwardsiella tarda, Streptococcus iniae, or red sea bream iridovirus (RSIV) exhibited significant upregulation of RbCTSA expression compared to the controls. Our results reveal that RbCTSA may be involved in the immune responses of rock bream.

Small Molecule and Lipid Binding Mechanisms of Sortilin and Vps10 Indicated in Altered Trafficking Patterns and Membrane Fusion

This study focuses on the regulation of sortilin/Vps10p‐directed protein trafficking. Human sortilin is a Vps10 domain‐containing transmembrane receptor that interacts with Apo B100‐containing VLDL, PCSK9 and LDL in the lumen of hepatic secretory vesicles. The Vps10 domain name comes from the yeast orthologue Vps10p, a carboxypeptidase Y (CPY) sorting receptor responsible for trafficking proteins to the vacuole in yeast. We have recently shown that vesicular VLDL contains phosphatidylinositol (3,4,5)‐trisphosphate (PIP3). Furthermore, we found that sortilin directly binds PIP3 using liposome binding assays as well as surface plasmon resonance using nanodiscs. We found that small molecules designed to bind human sortilin were also effective in yeast, leading to altered protein sorting to the yeast vacuole and secretion of CPY. Additional efforts using liposome floatation assays and microscale thermophoresis show that Vps10p and sortilin bind to additional membrane lipids. We hypothesize that differential binding different types of membrane lipids regulates Vps10‐controlled protein sorting. Further characterization of sortilin probes in yeast identified a variety of potential protein targets including the Rab7 orthologue Ypt7. This late endosomal GTPase is required for retrograde trafficking to the Golgi, anterograde traffic to the endolysosomal pathway, and vacuole homotypic fusion. Both deletion of VPS10 or binding of the receptor with sortilin‐targeting compounds led to defective Ypt7 trafficking to vacuoles and inhibited homotypic vacuole fusion. To complement direct binding studies, yeast Vps10p homologues have been identified and structural research performed to better understand binding features of Vps10‐like luminal domains. Elucidation of the relationship between specific luminal binding interactions of sorting receptors and vesicle trafficking predestination should allow better understanding of mechanistic interactions of yeast Vps10p with cytosolic trafficking factors and components.Support or Funding InformationNIGMS R01‐GM101132

Yeast dynamin Vps1 associates with clathrin to facilitate vesicular trafficking and controls Golgi homeostasis

The yeast dynamin Vps1 acts cooperatively with many proteins at diverse cellular locations for endocytosis, protein sorting, and membrane fusion and fission. It has been proposed that Vps1 is functionally linked to clathrin heavy chain 1 (Chc1), but the question of how, where, and when they function together remains unknown. Here we report that Vps1 arrives at the Golgi after clathrin, and that loss of Vps1 leads to a shift in the cellular localization of clathrin to the late endosome and vacuole, not vice versa. Our two-hybrid-based approach provides evidence that full-length Vps1 and its truncated versions bind to the C-terminal region of the Chc1. Cells lacking both Vps1 and Chc1 displayed more severe defects in carboxypeptidase Y (CPY) sorting at the Golgi than those in Vps1-deficient cells. Further, these Vps1 fragments became dominant-negative for CPY sorting upon overexpression. These results suggest that Vps1 binds to Chc1 and functions together at the Golgi for efficient Golgi-to-endosome membrane trafficking. In addition, we found that Vps1, without the aid of clathrin, plays a role in controlling the number and turnover of late Golgi.