Endosome Fusion Research Articles

Dynamic pH measurements of intracellular pathways using nano-plasmonic assemblies.

Plasmonic Ag nano-assemblies moving in a living cell were employed to visualize the spatiotemporal change of intracellular pH by surface-enhanced Raman scattering. Ag nano-assemblies were functionalized with 4-mercaptobenzoic acid (p-MBA) to monitor the variation of the surrounding pH. SERS spectra from the functionalized nano-assemblies that were transported in a cell were recorded to estimate the local pH on the pathways travelled by the nano-assemblies. 3D nanoparticle tracking and SERS measurements were simultaneously performed to trace the pH dynamics with a spatial accuracy of several tens of nanometers and a temporal resolution of 200 ms, which are sufficient to reveal pH changes associated with intracellular transportation. The pH trajectory of the functionalized nanoparticle can be used to visualize the dynamic change of the chemical environment caused by organelle interactions, such as endosomal trafficking and lysosomal fusion. This breakthrough creates a new paradigm for intracellular analysis using SERS and reveals new capability for rapid use of SERS in biological analysis.

Invariant chain regulates endosomal fusion and maturation through an interaction with the SNARE Vti1b.

The invariant chain (Ii, also known as CD74) is a multifunctional regulator of adaptive immune responses and is responsible for sorting major histocompatibility complex class I and class II (MHCI and MHCII, respectively) molecules, as well as other Ii-associated molecules, to a specific endosomal pathway. When Ii is expressed, endosomal maturation and proteolytic degradation of proteins are delayed and, in non-antigen presenting cells, the endosomal size increases, but the molecular mechanisms underlying this are not known. We identified that a SNARE, Vti1b, is essential for regulating these Ii-induced effects. Vti1b binds to Ii and is localized at the contact sites of fusing Ii-positive endosomes. Furthermore, truncated Ii lacking the cytoplasmic tail, which is not internalized from the plasma membrane, relocates Vti1b to the plasma membrane. Knockout of Ii in an antigen-presenting cell line was found to speed up endosomal maturation, whereas silencing of Vti1b inhibits the Ii-induced maturation delay. Our results suggest that Ii, by interacting with the SNARE Vti1b in antigen-presenting cells, directs specific Ii-associated SNARE-mediated fusion in the early part of the endosomal pathway that leads to a slower endosomal maturation for efficient antigen processing and MHC antigen loading.

Phosphatidylinositol 3,4-bisphosphate synthesis and turnover are spatially segregated in the endocytic pathway

Phosphatidylinositol 3,4-bisphosphate synthesis and turnover are spatially segregated in the endocytic pathway

Periodontal Disease in the COVID-19 Era: Potential Reservoir and Increased Risk for SARS-CoV-2

Since December 2019, an outbreak of Coronavirus Disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in China and is now becoming a global Pandemic, with over 10.5 million cases worldwide. Angiotensin-converting enzyme-2 (ACE-2) has been considered the main receptor for the SARS-CoV-2 entry into human cells, and they are known to be present not only in lungs (therefore the common viral pneumonia) but also in nasopharyngeal mucosa, salivary cells and oral epithelial cells. Moreover, there seems to be recent evidence showing that the crevicular fluid on the periodontal pockets of patients presenting periodontitis could harbor SARS-CoV-2 and act as a potential reservoir for increased viral load in the oral cavity of COVID-positive patients. Additionally, sites with active periodontal disease might contribute for virus binding and tissue infection due to elevated expression of furin and cathepsin L proteases, which play a major role in enabling the SARS-CoV-2 to bind ACE-2 receptors and facilitating endosomal fusion in the host cells. Taken together this news and views article highlight possible virus reservoirs in COVID-positive patients, as well as increased risk for infection in patients with active periodontal disease, further proposing potential targets for antiviral intervention on SARS-CoV-2 infection.

PmEEA1, the early endosomal protein is employed by YHV for successful infection in Penaeus monodon

Yellow head disease (YHD) is an infectious disease of Penaeus monodon which is caused by the yellow head virus (YHV). YHV infection invariably leads to 100% shrimp mortality within 3–5 days. Currently, an effective method to prevent or cure shrimp from YHV infection has not been elucidated. Therefore, the molecular mechanism underlying YHV infection should be examined. In this study, early endosome antigen 1 (EEA1) protein that was involved in the tethering step of the vesicle and early endosome fusion was investigated during YHV infection. The open reading frame of P. monodon EEA1 (PmEEA1) was cloned and sequenced (3000 bp). It encoded a putative protein of 999 amino acids and contained the zinc finger C2H2 domain signature at the N-terminus and the FYVE domain at the C-terminus. Suppression of PmEEA1 by specific dsRNA in shrimp showed inhibition of YHV replication after 48 h post YHV injection (hpi). On the other hand, shrimp received only NaCl without any dsRNA showed high YHV levels at approximately one hundred thousand times at 24 hpi and 48 hpi. Moreover, silencing of PmEEA1 by specific dsRNA followed by YHV challenge demonstrated a delay in shrimp mortality from 60 hpi to 168 hpi when compared to the control. These results indicated that YHV required PmEEA1 for trafficking within the infected cells, strongly suggesting that PmEEA1 may be a potential target to control and prevent YHV infection in P. monodon.

Structure and Dynamics of Mono- vs. Doubly Lipidated Rab5 in Membranes.

The Rab5 small GTPase is a regulator of endosomal trafficking and vesicle fusion. It possesses two adjacent cysteine residues for post-translational geranylgeranylation at its C-terminus for the protein to associate with the early endosome membrane. We compare the effect of mono-lipidification of only one cysteine residue with the doubly modified, fully functional Rab protein in both guanosine diphosphate (GDP)- and guanosine triphosphate (GTP)-bound states and in different membranes (one, three, and six-component membranes). Molecular simulations show that the mono-geranylgeranylated protein is less strongly associated with the membranes and diffuses faster than the doubly lipidated protein. The geranylgeranyl anchor membrane insertion depth is smaller and the protein–membrane distance distribution is broad and uncharacteristic for the membrane composition. The mono-geranylgeranylated protein reveals an unspecific association with the membrane and an orientation at the membrane that does not allow a nucleotide-specific recruitment of further effector proteins. This work shows that double-lipidification is critical for Rab5 to perform its physiological function and mono-geranylgeranylation renders it membrane-associated but non-functional.

UBAP1 mutations cause juvenile-onset hereditary spastic paraplegias (SPG80) and impair UBAP1 targeting to endosomes.

We aimed to find a new causative gene and elucidate the molecular mechanisms underlying a new type of hereditary spastic paraplegia (HSP). Patients with HSP were recruited from the Japan Spastic Paraplegia Research Consortium (JASPAC). Exome sequencing of genomic DNA from patients in four families was carried out, followed by Sanger sequencing of the UBAP1 gene. A mouse homolog of one UBAP1 frameshift mutation carried by one of the patients was created as a disease model. Functional properties of the UBAP1 wild type and UBAP1-mutant in mouse hippocampus neurons were examined. We identified three novel heterozygous loss of function mutations (c.425_426delAG, c.312delC, and c.535G>T) in the UBAP1 gene as the genetic cause of a new type of HSP (SPG80). All the patients presented identical clinical features of a pure type of juvenile-onset HSP. Functional studies on mouse hippocampal neurons revealed that the C-terminal deletion UBAP1-mutant of our disease model had lost its ability to bind ubiquitin in vitro. Overexpression of the UBAP1 wild type interacts directly with ubiquitin on enlarged endosomes, while the UBAP1-mutant cannot be recruited to endosome membranes. Our study demonstrated that mutations in the UBAP1 gene cause a new type of HSP and elucidated its pathogenesis. The full-length UBAP1 protein is involved in endosomal dynamics in neurons, while loss of UBAP1 function may perturb endosomal fusion and sorting of ubiquitinated cargos. These effects could be more prominent in neurons, thereby giving rise to the phenotype of a neurodegenerative disease such as HSP.

MCOLN1 Promotes Proliferation and Predicts Poor Survival of Patients with Pancreatic Ductal Adenocarcinoma.

Background MCOLN1 (mucolipin subfamily, member 1) was first identified as an autophagic regulator, which was essential for efficient fusion of both autophagosomes and late endosomes with lysosomes. This study is aimed at investigating the role of MCOLN1 in the development of pancreatic ductal adenocarcinoma (PDAC). Methods Immunohistochemistry (IHC) assay was conducted to evaluate the expression level of MCOLN1 in 82 human PDAC tumor tissues. Overall survival (OS) and recurrence-free survival (RFS) analysis was performed to assess the prognosis of patients. Colony formation and MTT assays [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide] were performed to measure the proliferation capacity of tumor cells. The expression level of related genes was measured by RT-PCR (reverse transcription polymerase chain reaction) and western blot assays. The animal model was used to examine the effects of indicated protein on tumorigenesis in vivo. Results The results of IHC showed that a high level of MCOLN1 expression was associated with the poor clinical characteristics of PDAC patients. OS and RFS were significantly worse in patients with high MCOLN1 expression. Silencing of MCOLN1 dramatically blocked the proliferation of PDAC cells. Mechanism studies confirmed that knockdown of MCOLN1 decreased the expression of Ki67 and PCNA (proliferating cell nuclear antigen), two markers of cell proliferation. In vivo, MCOILN1 depletion reduced the formation and growth of tumors in mice. Conclusion The high level of MCOLN1 expression was associated with poor clinical outcomes of PDAC patients. MCOLN1 ablation could inhibit PDAC proliferation of both in vitro and in vivo, which provide a new insight and novel therapeutic target for the treatment of PDAC.

Role of the RAB7 Protein in Tumor Progression and Cisplatin Chemoresistance.

RAB7 is a small guanosine triphosphatase (GTPase) extensively studied as regulator of vesicular trafficking. Indeed, its role is fundamental in several steps of the late endocytic pathway, including endosome maturation, transport from early endosomes to late endosomes and lysosomes, clustering and fusion of late endosomes and lysosomes in the perinuclear region and lysosomal biogenesis. Besides endocytosis, RAB7 is important for a number of other cellular processes among which, autophagy, apoptosis, signaling, and cell migration. Given the importance of RAB7 in these cellular processes, the interest to study the role of RAB7 in cancer progression is widely grown. Here, we describe the current understanding of oncogenic and oncosuppressor functions of RAB7 analyzing cellular context and other environmental factors in which it elicits pro and/or antitumorigenic effects. We also discuss the role of RAB7 in cisplatin resistance associated with its ability to regulate the late endosomal pathway, lysosomal biogenesis and extracellular vesicle secretion. Finally, we examined the potential cancer therapeutic strategies targeting the different molecular events in which RAB7 is involved.

Tamoxifen blocks retrograde trafficking of Shiga toxin 1 and 2 and protects against lethal toxicosis.

Shiga toxin 1 (STx1) and 2 (STx2), produced by Shiga toxin-producing Escherichia coli, cause lethal untreatable disease. The toxins invade cells via retrograde trafficking. Direct early endosome-to-Golgi transport allows the toxins to evade degradative late endosomes. Blocking toxin trafficking, particularly at the early endosome-to-Golgi step, is appealing, but transport mechanisms of the more disease-relevant STx2 are unclear. Using data from a genome-wide siRNA screen, we discovered that disruption of the fusion of late endosomes, but not autophagosomes, with lysosomes blocked the early endosome-to-Golgi transport of STx2. A subsequent screen of clinically approved lysosome-targeting drugs identified tamoxifen (TAM) to be a potent inhibitor of the trafficking and toxicity of STx1 and STx2 in cells. The protective effect was independent of estrogen receptors but dependent on the weak base property of TAM, which allowed TAM to increase endolysosomal pH and alter endosomal dynamics. Importantly, TAM treatment enhanced survival of mice injected with a lethal dose of STx1 or STx2. Thus, it may be possible to repurpose TAM for treating Shiga toxin-producing E. coli infections.

Proteasome 19S RP and translation preinitiation complexes are secreted within exosomes upon serum starvation.

The aim of our study was to investigate the impact of macroautophagy on exosome secretion. Exosomes are small membrane vesicles released in the extracellular space upon fusion of multivesicular endosomes with the plasma membrane. They were initially discovered as a way to remodel the reticulocyte plasma membrane before entering the blood circulation (Current Opinion in Hematology 2010, 17:177-183) and are now essentially studied as mediators of intercellular communication. Using iTRAQ proteomics, we compared the protein composition of purified exosomes secreted by cells impaired or not for macroautophagy by Atg5 depletion, during serum starvation conditions or complete medium culture. We show that the absence of serum modifies exosomal content, especially inducing secretion of two cytoplasmic protein complexes, namely proteasomal 19S regulatory particle (RP) and components of noncanonical translation preinitiation complex (PIC). This process is enhanced when autophagy is impaired by Atg5 depletion. Moreover, we show that the proteasome 20S core particle (CP) is released in the extracellular space. However, in striking contrast to what seen for its 19S RP regulator, release is independent of the exosomal vesicles, Atg5 expression and cell culture conditions. Exosome secretion can thus be considered as a cell process that participates in and reflects cell homeostasis, and care must be taken when studying potential extracellular function of exosomes due to the possible copurification of proteasome 20S CP.

Extracellular Vesicles: Exosomes and Microvesicles, Integrators of Homeostasis.

Extracellular vesicles (EVs), cell-derived membrane structures, are secreted after fusion of endosomes with the plasma membrane (exosomes) or shed from the plasma membrane (microvesicles). EVs play a key role both in physiological balance and homeostasis and in disease processes by their ability to participate in intercellular signaling and communication.

Vps11 and Vps18 of Vps-C membrane traffic complexes are E3 ubiquitin ligases and fine-tune signalling

In response to extracellular signals, many signalling proteins associated with the plasma membrane are sorted into endosomes. This involves endosomal fusion, which depends on the complexes HOPS and CORVET. Whether and how their subunits themselves modulate signal transduction is unknown. We show that Vps11 and Vps18 (Vps11/18), two common subunits of the HOPS/CORVET complexes, are E3 ubiquitin ligases. Upon overexpression of Vps11/Vps18, we find perturbations of ubiquitination in signal transduction pathways. We specifically demonstrate that Vps11/18 regulate several signalling factors and pathways, including Wnt, estrogen receptor α (ERα), and NFκB. For ERα, we demonstrate that the Vps11/18-mediated ubiquitination of the scaffold protein PELP1 impairs the activation of ERα by c-Src. Thus, proteins involved in membrane traffic, in addition to performing their well-described role in endosomal fusion, fine-tune signalling in several different ways, including through ubiquitination.

Characterization of putative proteins encoded by variable ORFs in white spot syndrome virus genome

BackgroundWhite Spot Syndrome Virus (WSSV) is an enveloped double-stranded DNA virus which causes mortality of several species of shrimp, being considered one of the main pathogens that affects global shrimp farming. This virus presents a complex genome of ~ 300 kb and viral isolates that present genomes with great identity. Despite this conservation, some variable regions in the WSSV genome occur in coding regions, and these putative proteins may have some relationship with viral adaptation and virulence mechanisms. Until now, the functions of these proteins were little studied. In this work, sequences and putative proteins encoded by WSSV variable regions were characterized in silico.ResultsThe in silico approach enabled determining the variability of some sequences, as well as the identification of some domains resembling the Formin homology 2, RNA recognition motif, Xeroderma pigmentosum group D repair helicase, Hemagglutinin and Ankyrin motif. The information obtained from the sequences and the analysis of secondary and tertiary structure models allow to infer that some of these proteins possibly have functions related to protein modulation/degradation, intracellular transport, recombination and endosome fusion events.ConclusionsThe bioinformatics approaches were efficient in generating three-dimensional models and to identify domains, thereby enabling to propose possible functions for the putative polypeptides produced by the ORFs wsv129, wsv178, wsv249, wsv463a, wsv477, wsv479, wsv492, and wsv497.

SUN-268 Severe Infantile Insulin Resistance: A Novel Feature of MIRAGE Syndrome

Background MIRAGE syndrome is a rare multisystem disorder characterized by myelodysplasia, infection, growth restriction, adrenal hypoplasia, genital phenotypes, and enteropathy. The syndrome is associated with mutations in Sterile Alpha Motif Domain Containing 9 (SAMD9) gene, encoding an endosome fusion facilitator protein with additional function in growth factor signaling. To date, there are no reported cases of MIRAGE syndrome associated with severe insulin resistance in infancy. Clinical Case The patient was a premature female infant born at 30 6/7 weeks gestation with a history of IUGR and brain abnormalities. Her hospital course was complicated by respiratory failure, impaired immune function, chronic diarrhea due to pancreatic exocrine deficiency, anemia, and primary adrenal insufficiency. Whole exome sequencing revealed a novel, de novo missense mutation in SAMD9 (R1293Q), confirming the clinical diagnosis of MIRAGE syndrome. At 5 months of age, she developed severe hyperglycemia with insulin resistance (insulin 303 µIU/mL, normal < 13 µIU/mL, and C-peptide 18.2 ng/mL, normal < 2.2 ng/mL). Markers of insulin receptor defects were normal, including adiponectin, IGFBP-1, and SHBG, and no mutations were identified in INSR by whole exome sequencing. The patient had no episodes of diabetic ketosis or metabolic acidosis, and had no lipodystrophy or acanthosis nigricans on exam. Initially, an insulin infusion of 0.1-0.2 U/kg/hr was sufficient to maintain euglycemia. By age 6 months, persistent hyperglycemia led to a rapid increase of the insulin infusion to 18 U/kg/hr over the course of 3 days. On day 2 of insulin up titration, glucophage was started at 15 mg/kg/day. After 72 hours of escalating insulin doses and 48 hours of glucophage, both her hyperglycemia and insulin resistance rapidly improved, with insulin requirements decreasing to < 0.5 U/kg/hr over the next 2 days to maintain glucose in the 100-200 mg/dL range. In the following months, her insulin requirement varied greatly. While fasting, no insulin was needed to maintain euglycemia. However, while on parental nutrition or enteral feeds, insulin infusion requirement varied at 0.03 U/kg/hr - 0.5 U/kg/hr. At 8 months, she transitioned to subcutaneous insulin while enteral feeds were optimized and condensed. Subcutaneous insulin doses were gradually increased to 20-24 U/kg/day in an effort to maintain blood glucose <200 mg/dl. Unfortunately, at 9 months of age, the patient expired from sepsis. Conclusion This is the first case of severe insulin resistance presenting as a manifestation of MIRAGE syndrome due to a missense mutation in SAMD9. This patient had improvement in glycemic control after treatment with glucophage and insulin. SAMD9 mutations should be considered in patients with multiple endocrinopathies including adrenal hypoplasia, severe insulin resistance, and pancreatic exocrine deficiency.

Dysregulation of Microglial Function Contributes to Neuronal Impairment in Mcoln1a-Deficient Zebrafish

SummaryType IV mucolipidosis (ML-IV) is a neurodegenerative lysosome storage disorder caused by mutations in the MCOLN1 gene. However, the cellular and molecular bases underlying the neuronal phenotypes of ML-IV disease remain elusive. Using a forward genetic screening, we identified a zebrafish mutant, biluo, that harbors a hypomorphic mutation in mcoln1a, one of the two zebrafish homologs of mammalian MCOLN1. The mcoln1a-deficient mutants display phenotypes partially recapitulating the key features of ML-IV disorder, including the accumulation of enlarged late endosomes in microglia and aberrant neuronal activities in both spontaneous and visual-evoking conditions in optic tectal neurons. We further show that the accumulation of enlarged late endosomes in microglia is caused by the impairment of late endosome and lysosome fusion and the aberrant neuronal activities can be partially rescued by the reconstitution of Mcoln1a function in microglia. Our findings suggest that dysregulation of microglial function may contribute to the development and progression of ML-IV disease.

Exosomal release through TRPML1-mediated lysosomal exocytosis is required for adipogenesis

The lysosomal Ca2+ permeable channel TRPML1 (MCOLN1) plays key roles in lysosomal membrane trafficking, including the fusion of late endosomes to lysosomes and lysosomal exocytosis, both of which are essential for release of exosomes into the extracellular milieu. Multiple lines of evidence indicate that the contents of adipocyte-derived exosomes mediate diverse cellular responses, including adipogenic differentiation. In this study, we aimed to define the potential roles of TRPML1 in lysosomal membrane trafficking during adipogenesis and in exosomal release. In response to adipogenic stimuli, the endogenous TRPML1 expression in OP9 pre-adipocytes was increased in a time-dependent manner, and the acute deletion of TRPML1 reduced lipid synthesis and expression of differentiation-related marker genes. Notably, mature adipocyte-derived exosomes were found to be necessary for adipogenesis and were dependent on TRPML1-mediated lysosomal exocytosis. Taken together, our findings indicate that TRPML1 mediates diverse roles in adipocyte differentiation and exosomal release. Further, we propose that TRPML1 should be considered as a regulator of obesity-related diseases.

Rab5-independent activation and function of yeast Rab7-like protein, Ypt7p, in the AP-3 pathway.

The small GTPases, Rab5 and Rab7, are key regulators at multiple stages of the endocytic/endolysosomal pathway, including fusion and maturation of endosomes. In yeast, Vps21p (Rab5 homolog) recruits a GEF for Rab7 and activates the downstream Ypt7p (Rab7 homolog) on endosomal membrane. Although the model of this sequential activation from Vps21p to Ypt7p in the endocytic pathway has been established, activation mechanism of Ypt7p in the Vps21p-independent pathway has not been completely clarified. Here we show that Ypt7p is activated and mediates vacuolar fusion in cells lacking all yeast Rab5 genes, VPS21, YPT52, and YPT53. We also demonstrate that deletion of both VPS21 and YPT7 genes cause severe defect in the AP-3 pathway as well as the CPY pathway although the AP-3 pathway is mostly intact in each vps21Δ or ypt7Δ mutant. Interestingly, in vps21Δ ypt7Δ mutant cargos trafficked via the VPS or endocytic pathway accumulate beside nucleus whereas cargo trafficked via the AP-3 pathway disperse in the cytosol. These findings suggest that Ypt7p is activated and plays a Rab5-independent role in the AP-3-mediated pathway.

Mutations in Influenza A Virus Neuraminidase and Hemagglutinin Confer Resistance against a Broadly Neutralizing Hemagglutinin Stem Antibody.

Influenza A virus (IAV), a major cause of human morbidity and mortality, continuously evolves in response to selective pressures. Stem-directed, broadly neutralizing antibodies (sBnAbs) targeting the influenza virus hemagglutinin (HA) are a promising therapeutic strategy, but neutralization escape mutants can develop. We used an integrated approach combining viral passaging, deep sequencing, and protein structural analyses to define escape mutations and mechanisms of neutralization escape in vitro for the F10 sBnAb. IAV was propagated with escalating concentrations of F10 over serial passages in cultured cells to select for escape mutations. Viral sequence analysis revealed three mutations in HA and one in neuraminidase (NA). Introduction of these specific mutations into IAV through reverse genetics confirmed their roles in resistance to F10. Structural analyses revealed that the selected HA mutations (S123G, N460S, and N203V) are away from the F10 epitope but may indirectly impact influenza virus receptor binding, endosomal fusion, or budding. The NA mutation E329K, which was previously identified to be associated with antibody escape, affects the active site of NA, highlighting the importance of the balance between HA and NA function for viral survival. Thus, whole-genome population sequencing enables the identification of viral resistance mutations responding to antibody-induced selective pressure.IMPORTANCE Influenza A virus is a public health threat for which currently available vaccines are not always effective. Broadly neutralizing antibodies that bind to the highly conserved stem region of the influenza virus hemagglutinin (HA) can neutralize many influenza virus strains. To understand how influenza virus can become resistant or escape such antibodies, we propagated influenza A virus in vitro with escalating concentrations of antibody and analyzed viral populations by whole-genome sequencing. We identified HA mutations near and distal to the antibody binding epitope that conferred resistance to antibody neutralization. Additionally, we identified a neuraminidase (NA) mutation that allowed the virus to grow in the presence of high concentrations of the antibody. Virus carrying dual mutations in HA and NA also grew under high antibody concentrations. We show that NA mutations mediate the escape of neutralization by antibodies against HA, highlighting the importance of a balance between HA and NA for optimal virus function.

HOPS-dependent endosomal fusion required for efficient cytosolic delivery of therapeutic peptides and small proteins

Protein therapeutics represent a significant and growing component of the modern pharmacopeia, but their potential to treat human disease is limited because most proteins fail to traffic across biological membranes. Recently, we discovered a class of cell-permeant miniature proteins (CPMPs) containing a precisely defined, penta-arginine (penta-Arg) motif that traffics readily to the cytosol and nucleus of mammalian cells with efficiencies that rival those of hydrocarbon-stapled peptides active in animals and man. Like many cell-penetrating peptides (CPPs), CPMPs enter the endocytic pathway; the difference is that CPMPs containing a penta-Arg motif are released efficiently from endosomes, while other CPPs are not. Here, we seek to understand how CPMPs traffic from endosomes into the cytosol and what factors contribute to the efficiency of endosomal release. First, using two complementary cell-based assays, we exclude endosomal rupture as the primary means of endosomal escape. Next, using an RNA interference screen, fluorescence correlation spectroscopy, and confocal imaging, we identify VPS39-a gene encoding a subunit of the homotypic fusion and protein-sorting (HOPS) complex-as a critical determinant in the trafficking of CPMPs and hydrocarbon-stapled peptides to the cytosol. Although CPMPs neither inhibit nor activate HOPS function, HOPS activity is essential to efficiently deliver CPMPs to the cytosol. CPMPs localize within the lumen of Rab7+ and Lamp1+ endosomes and their transport requires HOPS activity. Overall, our results identify Lamp1+ late endosomes and lysosomes as portals for passing proteins into the cytosol and suggest that this environment is prerequisite for endosomal escape.