Late Endocytic Trafficking Research Articles

Sphingosine Kinases Promote Ebola Virus Infection and Can Be Targeted to Inhibit Filoviruses, Coronaviruses, and Arenaviruses Using Late Endocytic Trafficking to Enter Cells.

Entry of enveloped viruses in host cells requires the fusion of viral and host cell membranes, a process that is facilitated by viral fusion proteins protruding from the viral envelope. These viral fusion proteins need to be triggered by host factors, and for some viruses, this event occurs inside endosomes and/or lysosomes. Consequently, these 'late-penetrating viruses' must be internalized and delivered to entry-conducive intracellular vesicles. Because endocytosis and vesicular trafficking are tightly regulated cellular processes, late-penetrating viruses also depend on specific host proteins for efficient delivery to the site of fusion, suggesting that these could be targeted for antiviral therapy. In this study, we investigated a role for sphingosine kinases (SKs) in viral entry and found that chemical inhibition of sphingosine kinase 1 (SK1) and/or SK2 and knockdown of SK1/2 inhibited entry of Ebola virus (EBOV) into host cells. Mechanistically, inhibition of SK1/2 prevented EBOV from reaching late-endosomes and lysosomes that contain the EBOV receptor, Niemann Pick C1 (NPC1). Furthermore, we present evidence that suggests that the trafficking defect caused by SK1/2 inhibition occurs independently of sphingosine-1-phosphate (S1P) signaling through cell-surface S1P receptors. Lastly, we found that chemical inhibition of SK1/2 prevents entry of other late-penetrating viruses, including arenaviruses and coronaviruses, and inhibits infection by replication-competent EBOV and SARS-CoV-2 in Huh7.5 cells. In sum, our results highlight an important role played by SK1/2 in endocytic trafficking, which can be targeted to inhibit entry of late-penetrating viruses and could serve as a starting point for the development of broad-spectrum antiviral therapeutics.

Autophagy and Lysosomal Functionality in CMT2B Fibroblasts Carrying the RAB7K126R Mutation.

Charcot-Marie-Tooth type 2B (CMT2B) disease is a dominant axonal peripheral neuropathy caused by five mutations in the RAB7A gene. Autophagy and late endocytic trafficking were already characterized in CMT2B. Indeed, impairment of autophagy and an increase in lysosomal degradative activity were found in cells expressing the mutant proteins. Recently, we described a novel RAB7 mutation associated with predominantly motor CMT2 and impaired EGFR trafficking. With the aim to analyze the autophagy process and lysosomal activity in CMT2B fibroblasts carrying the p.K126R RAB7 novel mutation and to investigate further the causes of the different phenotype, we have performed Western blot, immunofluorescence and cytometric analyses monitoring autophagic markers and endocytic proteins. Moreover, we investigated lipophagy by analyzing accumulation of lipid droplets and their co-localization with endolysosomal degradative compartments. We found that cells expressing the RAB7K126R mutant protein were characterized by impairment of autophagy and lipophagy processes and by a moderate increase in lysosomal activity compared to the previously studied cells carrying the RAB7V162M mutation. Thus, we concluded that EGFR trafficking alterations and a moderate increase in lysosomal activity with concomitant impairment of autophagy could induce the specific predominantly motor phenotype observed in K126R patients.

C5orf51 is a component of the MON1-CCZ1 complex and controls RAB7A localization and stability during mitophagy

ABSTRACT Depolarized mitochondria can be degraded via mitophagy, a selective form of autophagy. The RAB GTPase RAB7A was recently shown to play a key role in this process. RAB7A regulates late endocytic trafficking under normal growth conditions but is translocated to the mitochondrial surface following depolarization. However, how RAB7A activity is regulated during mitophagy is not understood. Here, using a proximity-dependent biotinylation approach (miniTurbo), we identified C5orf51 as a specific interactor of GDP-locked RAB7A. C5orf51 also interacts with the RAB7A guanine nucleotide exchange factor (GEF) complex members MON1 and CCZ1. In the absence of C5orf51, localization of RAB7A on depolarized mitochondria is compromised and the protein is degraded by the proteasome. Furthermore, depletion of C5orf51 also inhibited ATG9A recruitment to depolarized mitochondria. Together, these results indicate that C5orf51 is a positive regulator of RAB7A in its shuttling between late endosomes and mitochondria to enable mitophagy. Abbreviations: ATG9A: autophagy related 9A; Baf A1: bafilomycin A1; BioID: proximity-dependent biotin identification; CCCP: carbonyl cyanide m-chlorophenylhydrazone; CCZ1: CCZ1 homolog, vacuolar protein trafficking and biogenesis associated; DQ-BSA: dye quenched-bovine serum albumin; FYCO1: FYVE and coiled-coil domain autophagy adaptor 1; GAP: GTPase activating protein; GEF: guanine nucleotide exchange factor; KO: knockout; LRPPRC: leucine rich pentatricopeptide repeat containing; MG132: carbobenzoxy-Leu-Leu-leucinal; MON1: MON1 homolog, secretory trafficking associated; mtDNA: mitochondrial DNA; PINK1: PTEN induced kinase 1; PRKN/PARKIN: parkin RBR E3 ubiquitin protein ligase; RMC1: regulator of MON1-CCZ1; TBC1D15: TBC1 domain family member 15; TBC1D17: TBC1 domain family member 17; TOMM20: translocase of outer mitochondrial membrane 20; WDR91: WD repeat domain 91; WT: wild type.

Emerging Roles of Small GTPases in Islet β-Cell Function.

Several small guanosine triphosphatases (GTPases) from the Ras protein superfamily regulate glucose-stimulated insulin secretion in the pancreatic islet β-cell. The Rho family GTPases Cdc42 and Rac1 are primarily involved in relaying key signals in several cellular functions, including vesicle trafficking, plasma membrane homeostasis, and cytoskeletal dynamics. They orchestrate specific changes at each spatiotemporal region within the β-cell by coordinating with signal transducers, guanine nucleotide exchange factors (GEFs), GTPase-activating factors (GAPs), and their effectors. The Arf family of small GTPases is involved in vesicular trafficking (exocytosis and endocytosis) and actin cytoskeletal dynamics. Rab-GTPases regulate pre-exocytotic and late endocytic membrane trafficking events in β-cells. Several additional functions for small GTPases include regulating transcription factor activity and mitochondrial dynamics. Importantly, defects in several of these GTPases have been found associated with type 2 diabetes (T2D) etiology. The purpose of this review is to systematically denote the identities and molecular mechanistic steps in the glucose-stimulated insulin secretion pathway that leads to the normal release of insulin. We will also note newly identified defects in these GTPases and their corresponding regulatory factors (e.g., GDP dissociation inhibitors (GDIs), GEFs, and GAPs) in the pancreatic β-cells, which contribute to the dysregulation of metabolism and the development of T2D.

VPS18-regulated vesicle trafficking controls the secretion of pectin and its modifying enzyme during pollen tube growth in Arabidopsis.

In eukaryotes, homotypic fusion and vacuolar protein sorting (HOPS) as well as class C core vacuole/endosome tethering (CORVET) are evolutionarily conserved membrane tethering complexes that play important roles in lysosomal/vacuolar trafficking. Whether HOPS and CORVET control endomembrane trafficking in pollen tubes, the fastest growing plant cells, remains largely elusive. In this study, we demonstrate that the four core components shared by the two complexes, Vacuole protein sorting 11 (VPS11), VPS16, VPS33, and VPS18, are all essential for pollen tube growth in Arabidopsis thaliana and thus for plant reproduction success. We used VPS18 as a representative core component of the complexes to show that the protein is localized to both multivesicular bodies (MVBs) and the tonoplast in a growing pollen tube. Mutant vps18 pollen tubes grew more slowly in vivo, resulting in a significant reduction in male transmission efficiency. Additional studies revealed that membrane fusion from MVBs to vacuoles is severely compromised in vps18 pollen tubes, corroborating the function of VPS18 in late endocytic trafficking. Furthermore, vps18 pollen tubes produce excessive exocytic vesicles at the apical zone and excessive amounts of pectin and pectin methylesterases in the cell wall. In conclusion, this study establishes an additional conserved role of HOPS/CORVET in homotypic membrane fusion during vacuole biogenesis in pollen tubes and reveals a feedback regulation of HOPS/CORVET in the secretion of cell wall modification enzymes of rapidly growing plant cells.

Knockdown of Rab7a suppresses the proliferation, migration, and xenograft tumor growth of breast cancer cells.

Breast cancer is a common invasive cancer in women. Ras-related protein Rab-7a (Rab7a) is involved in late endocytic trafficking, while its role in breast cancer is largely unclear. In the present study, we investigated the role of Rab7a in breast cancer. Comparing with adjacent breast tissues, Rab7a expression was increased in breast cancer tissues. Using lentivirus-mediated knockdown strategy, we found that Rab7a silencing inhibited the proliferation and colony formation of MDA-MB-231 cells. Apoptosis and G2 cell cycle arrest were induced in Rab7a knockdown. By contrast, Rab7a suppressed the apoptosis and promoted proliferation and colony formation of MCF-7 cells. The migration of MDA-MB-231 cells was suppressed by Rab7a knockdown. In vivo, depletion of Rab7a inhibited the xenograft tumor development of MDA-MB-231 cells. Altogether, our results highlight the novel function of Rab7a in the proliferation, invasion, and xenograft tumor development of breast cancer cells.

Modulation of RAB7A Protein Expression Determines Resistance to Cisplatin through Late Endocytic Pathway Impairment and Extracellular Vesicular Secretion.

Background: Cisplatin (CDDP) is widely used in treatment of cancer, yet patients often develop resistance with consequent therapeutical failure. In CDDP-resistant cells alterations of endocytosis and lysosomal functionality have been revealed, although their causes and contribution to therapy response are unclear. Methods: We investigated the role of RAB7A, a key regulator of late endocytic trafficking, in CDDP-resistance by comparing resistant and sensitive cells using western blotting, confocal microscopy and real time PCR. Modulation of RAB7A expression was performed by transfection and RNA interference, while CDDP sensitivity and intracellular accumulation were evaluated by viability assays and chemical approaches, respectively. Also extracellular vesicles were purified and analyzed. Finally, correlations between RAB7A and chemotherapy response was investigated in human patient samples. Results: We demonstrated that down-regulation of RAB7A characterizes the chemoresistant phenotype, and that RAB7A depletion increases CDDP-resistance while RAB7A overexpression decreases it. In addition, increased production of extracellular vesicles is modulated by RAB7A expression levels and correlates with reduction of CDDP intracellular accumulation. Conclusions: We demonstrated, for the first time, that RAB7A regulates CDDP resistance determining alterations in late endocytic trafficking and drug efflux through extracellular vesicles.

Alterations of autophagy in the peripheral neuropathy Charcot-Marie-Tooth type 2B

ABSTRACTCharcot-Marie-Tooth type 2B (CMT2B) disease is a dominant axonal peripheral neuropathy caused by 5 mutations in the RAB7A gene, a ubiquitously expressed GTPase controlling late endocytic trafficking. In neurons, RAB7A also controls neuronal-specific processes such as NTF (neurotrophin) trafficking and signaling, neurite outgrowth and neuronal migration. Given the involvement of macroautophagy/autophagy in several neurodegenerative diseases and considering that RAB7A is fundamental for autophagosome maturation, we investigated whether CMT2B-causing mutants affect the ability of this gene to regulate autophagy. In HeLa cells, we observed a reduced localization of all CMT2B-causing RAB7A mutants on autophagic compartments. Furthermore, compared to expression of RAB7AWT, expression of these mutants caused a reduced autophagic flux, similar to what happens in cells expressing the dominant negative RAB7AT22N mutant. Consistently, both basal and starvation-induced autophagy were strongly inhibited in skin fibroblasts from a CMT2B patient carrying the RAB7AV162M mutation, suggesting that alteration of the autophagic flux could be responsible for neurodegeneration.

Sequential actions of phosphatidylinositol phosphates regulate phagosome-lysosome fusion.

Phagosomes mature into phagolysosomes by sequential fusion with early endosomes, late endosomes, and lysosomes. Phagosome-with-lysosome fusion (PLF) results in the delivery of lysosomal hydrolases into phagosomes and in digestion of the cargo. The machinery that drives PLF has been little investigated. Using a cell-free system, we recently identified the phosphoinositide lipids (PIPs) phosphatidylinositol 3-phosphate (PI(3)P) and phosphatidylinositol 4-phosphate (PI(4)P) as regulators of PLF. We now report the identification and the PIP requirements of four distinct subreactions of PLF. Our data show that (i) PI(3)P and PI(4)P are dispensable for the disassembly and activation of (phago)lysosomal soluble N-ethylmaleimide-sensitive factor attachment protein receptors, that (ii) PI(3)P is required only after the tethering step, and that (iii) PI(4)P is required during and after tethering. Moreover, our data indicate that PI(4)P is needed to anchor Arl8 (Arf-like GTPase 8) and its effector homotypic fusion/vacuole protein sorting complex (HOPS) to (phago)lysosome membranes, whereas PI(3)P is required for membrane association of HOPS only. Our study provides a first link between PIPs and established regulators of membrane fusion in late endocytic trafficking.

Rab7a regulates cell migration through Rac1 and vimentin

Rab7a, a small GTPase of the Rab family, is localized to late endosomes and controls late endocytic trafficking. The discovery of several Rab7a interacting proteins revealed that Rab7a function is closely connected to cytoskeletal elements. Indeed, Rab7a recruits on vesicles RILP and FYCO that are responsible for the movement of Rab7a-positive vesicles and/or organelles on microtubule tracks, but also directly interacts with Rac1, a fundamental regulator of actin cytoskeleton, and with peripherin and vimentin, two intermediate filament proteins. Considering all these interactions and, in particular, the fact that Rac1 and vimentin are key factors for cellular motility, we investigated a possible role of Rab7a in cell migration. We show here that Rab7a is needed for cell migration as Rab7a depletion causes slower migration of NCI H1299 cells affecting cell velocity and directness. Rab7a depletion negatively affects adhesion and spreading onto fibronectin substrates, altering β1-integrin activation, localization and intracellular trafficking, and myosin X localization. In fact, Rab7a-depleted cells show 40% less filopodia and active integrin accumulates at the leading edge of migrating cells. Furthermore, Rab7a depletion decreases the amount of active Rac1 but not its abundance and reduces the number of cells with vimentin filaments facing the wound, indicating that Rab7a has a role in the orientation of vimentin filaments during migration. In conclusion, our results demonstrate a key role of Rab7a in the regulation of different aspects of cell migration.

PTEN modulates EGFR late endocytic trafficking and degradation by dephosphorylating Rab7.

Tumour suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a lipid phosphatase that negatively regulates growth factor-induced survival signalling. Here, we demonstrate that PTEN attenuates epidermal growth factor receptor (EGFR) signalling by promoting late endosome maturation by virtue of its protein phosphatase activity. Loss of PTEN impairs the transition of ligand-bound EGFR from early to late endosomes. We unveil Rab7, a critical GTPase for endosome maturation, as a functional PTEN interacting partner. PTEN dephosphorylates Rab7 on two conserved residues S72 and Y183, which are necessary for GDP dissociation inhibitor (GDI)-dependent recruitment of Rab7 on to late endosomes and subsequent maturation. Thus, our findings reveal PTEN-dependent endosome maturation through phosphoregulation of Rab7 as an important route of controlling EGFR signalling.

Vps34 regulates Rab7 and late endocytic trafficking through recruitment of the GTPase-activating protein Armus.

The class III phosphoinositide 3-kinase (PI3K) Vps34 (also known as PIK3C3 in mammals) produces phosphatidylinositol 3-phosphate [PI(3)P] on both early and late endosome membranes to control membrane dynamics. We used Vps34-deficient cells to delineate whether Vps34 has additional roles in endocytic trafficking. In Vps34-/- mouse embryonic fibroblasts (MEFs), transferrin recycling and EEA1 membrane localization were unaffected despite elevated Rab5-GTP levels. Strikingly, a large increase in Rab7-GTP levels, an accumulation of enlarged late endosomes, and decreased EGFR degradation were observed in Vps34-deficient cells. The hyperactivation of Rab7 in Vps34-deficient cells stemmed from the failure to recruit the Rab7 GTPase-activating protein (GAP) Armus (also known as TBC1D2), which binds to PI(3)P, to late endosomes. Protein-lipid overlay and liposome-binding assays reveal that the putative pleckstrin homology (PH) domain in Armus can directly bind to PI(3)P. Elevated Rab7-GTP led to the failure of intraluminal vesicle (ILV) formation and lysosomal maturation. Rab7 silencing and Armus overexpression alleviated the vacuolization seen in Vps34-deficient cells. Taken together, these results demonstrate that Vps34 has a previously unknown role in regulating Rab7 activity and late endosomal trafficking.

A moving view: subcellular trafficking processes in pattern recognition receptor-triggered plant immunity.

A significant challenge for plants is to induce localized defense responses at sites of pathogen attack. Therefore, host subcellular trafficking processes enable accumulation and exchange of defense compounds, which contributes to the plant on-site defenses in response to pathogen perception. This review summarizes our current understanding of the transport processes that facilitate immunity, the significance of which is highlighted by pathogens reprogramming membrane trafficking through host cell translocated effectors. Prominent immune-related cargos of plant trafficking pathways are the pattern recognition receptors (PRRs), which must be present at the plasma membrane to sense microbes in the apoplast. We focus on the dynamic localization of the FLS2 receptor and discuss the pathways that regulate receptor transport within the cell and their link to FLS2-mediated immunity. One emerging theme is that ligand-induced late endocytic trafficking is conserved across different PRR protein families as well as across different plant species.

Alterations in late endocytic trafficking related to the pathobiology of LRRK2-linked Parkinson's disease.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene comprise the most common cause of familial Parkinson's disease (PD), and variants increase the risk for sporadic PD. LRRK2 displays kinase and GTPase activity, and altered catalytic activity correlates with neurotoxicity, making LRRK2 a promising therapeutic target. Despite the importance of LRRK2 for disease pathogenesis, its normal cellular function, and the mechanism(s) by which pathogenic mutations cause neurodegeneration remain unclear. LRRK2 seems to regulate a variety of intracellular vesicular trafficking events to and from the late endosome in a manner dependent on various Rab proteins. At least some of those events are further regulated by LRRK2 in a manner dependent on two-pore channels (TPCs). TPCs are ionic channels localized to distinct endosomal structures and can cause localized calcium release from those acidic stores, with downstream effects on vesicular trafficking. Here, we review current knowledge about the link between LRRK2, TPC- and Rab-mediated vesicular trafficking to and from the late endosome, highlighting a possible cross-talk between endolysosomal calcium stores and Rab proteins underlying pathomechanism(s) in LRRK2-related PD.

Rerouting of plant late endocytic trafficking toward a pathogen interface.

A number of plant pathogenic and symbiotic microbes produce specialized cellular structures that invade host cells where they remain enveloped by host-derived membranes. The mechanisms underlying the biogenesis and functions of host-microbe interfaces are poorly understood. Here, we show that plant late endocytic trafficking is diverted toward the extrahaustorial membrane (EHM); a host-pathogen interface that develops in plant cells invaded by Irish potato famine pathogen Phytophthora infestans. A late endosome and tonoplast marker protein Rab7 GTPase RabG3c, but not a tonoplast-localized sucrose transporter, is recruited to the EHM, suggesting specific rerouting of vacuole-targeted late endosomes to a host-pathogen interface. We revealed the dynamic nature of this process by showing that, upon activation, a cell surface immune receptor traffics toward the haustorial interface. Our work provides insight into the biogenesis of the EHM and reveals dynamic processes that recruit membrane compartments and immune receptors to this host-pathogen interface.

Rab7a: the master regulator of vesicular trafficking

The membrane flow of eukaryotic cells occurs through vesicles that bud from a donor compartment, move and fuse with an acceptor compartment. Rab (Ras-related in brain), which belong to the Ras superfamily of small GTPases, emerged as a central player of vesicle mobility in both secretory and endocytic pathway, Rab7a being a master regulator of late endocytic trafficking. Elucidation of how mutant or dysregulated Rab7 GTPase and accessory proteins contribute to organ specific and systemic disease remains an area of intensive study and an essential foundation for effective drug targeting. Mutation of Rab7 or associated regulatory proteins causes numerous human genetic diseases. Cancer and neurodegeneration represent examples of acquired human diseases resulting from the up- or down-regulation or aberrant function of Rab7. The broad range of physiologic processes affected by altered Rab7 activity is based on its pivotal roles in membrane trafficking and signaling. The Rab7-regulated processes of cargo sorting, cytoskeletal translocation of vesicles and appropriate docking and fusion with the target membranes control cell metabolism, growth and differentiation. In this review, role of Rab7 in endocytosis is evaluated to illustrate normal function and the consequences of dysregulation resulting in human disease. Selected examples are designed to illustrate how defects in Rab7 activity alter endocytic trafficking that underlie neurologic, lipid storage, and bone disorders as well as cancer. Biomedical Reviews 2014; 25: 67-81.

Snapin associates with late endocytic compartments and interacts with late endosomal SNAREs

Late endocytic membrane trafficking delivers target materials and newly synthesized hydrolases into lysosomes and is critical for maintaining an efficient degradation process and cellular homoeostasis. Although some features of late endosome-lysosome trafficking have been described, the mechanisms underlying regulation of this event remain to be elucidated. Our previous studies showed that Snapin, as a SNAP25 (25 kDa synaptosome-associated protein)-binding protein, plays a critical role in priming synaptic vesicles for synchronized fusion in neurons. In the present study, we report that Snapin also associates with late endocytic membranous organelles and interacts with the late endosome-targeted SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) complex. Using a genetic mouse model, we further discovered that Snapin is required to maintain a proper balance of the late endocytic protein LAMP-1 (lysosome-associated membrane protein-1) and late endosomal SNARE proteins syntaxin 8 and Vti1b (vesicle transport through interaction with target SNAREs homologue 1b). Deleting the snapin gene in mice selectively led to the accumulation of these proteins in late endocytic organelles. Thus our present study suggests that Snapin serves as an important regulator of the late endocytic fusion machinery, in addition to its established role in regulating synaptic vesicle fusion.

Rab7 Regulates Late Endocytic Trafficking Downstream of Multivesicular Body Biogenesis and Cargo Sequestration

The small molecular weight G-protein RAB7 is localized to both early and late endosomes and has been shown to be critical for trafficking through the endocytic pathway. The role of RAB7 in the endocytic pathway has been controversial, with some groups reporting that it regulates trafficking from early to late endosomes and others ascribing its role to trafficking between late endosomes and lysosomes. In this study, we use RNA interference to identify the exact step RAB7 regulates in the movement of the epidermal growth factor receptor (EGFR) from the cell surface to the lysosome. In the absence of RAB7, trafficking of the EGF.EGFR complex through the early endosome to the late endosome/multivesicular body (LE/MVB) does not change, but exiting from the LE/MVB is blocked. Ultrastructural analysis reveals that RAB7 is not required for formation of intraluminal vesicles of the LE/MVB, since RAB7-deficient cells have an increased number of enlarged LE/MVBs densely packed with intraluminal vesicles. Biochemical data indicate that the EGFR complex is sequestered in these intraluminal vesicles. Together, these data provide evidence that RAB7 is required for the transfer of cargo from the LE/MVB to the lysosome and for endocytic organelle maintenance.

Densovirus Infectious Pathway Requires Clathrin-Mediated Endocytosis Followed by Trafficking to the Nucleus

Junonia coenia densovirus (JcDNV) is an ambisense insect parvovirus highly pathogenic for lepidopteran pests at larval stages. The potential use of DNVs as biological control agents prompted us to reinvestigate the host range and cellular mechanisms of infection. In order to understand the early events of infection, we set up a functional infection assay in a cell line of the pest Lymantria dispar to determine the intracellular pathway undertaken by JcDNV to infect a permissive lepidopteran cell line. Our results show that JcDNV particles are rapidly internalized into clathrin-coated vesicles and slowly traffic within early and late endocytic compartments. Blocking late-endocytic trafficking or neutralizing the pH with drugs inhibited infection. During internalization, disruption of the cytoskeleton, and inhibition of phosphatidylinositol 3-kinase blocked the movement of vesicles containing the virus to the nucleus and impaired infection. In summary, our results define for the first time the early endocytic steps required for a productive DNV infection.

An analysis of the role of a retroendocytosis pathway in ABCA1-mediated cholesterol efflux from macrophages

The ATP binding cassette transporter A-1 (ABCA1) is critical for apolipoprotein-mediated cholesterol efflux, an important mechanism employed by macrophages to avoid becoming lipid-laden foam cells, the hallmark of early atherosclerotic lesions. It has been proposed that lipid-free apolipoprotein A-I (apoA-I) enters the cell and is resecreted as a lipidated particle via a retroendocytosis pathway during ABCA1-mediated cholesterol efflux from macrophages. To determine the functional importance of such a pathway, confocal microscopy was used to characterize the internalization of a fully functional apoA-I cysteine mutant containing a thiol-reactive fluorescent probe in cultured macrophages. ApoA-I was also endogenously labeled with (35)S-methionine to quantify cellular uptake and to determine the metabolic fate of the internalized protein. It was found that apoA-I was specifically taken inside macrophages and that a small amount of intact apoA-I was resecreted from the cells. However, a majority of the label that reappeared in the media was degraded. We estimate that the mass of apoA-I retroendocytosed is not sufficient to account for the HDL produced by the cholesterol efflux reaction. Furthermore, we have demonstrated that lipid-free apoA-I-mediated cholesterol efflux from macrophages can be pharmacologically uncoupled from apoA-I internalization into cells. On the basis these findings, we present a model in which the ABCA1-mediated lipid transfer process occurs primarily at the membrane surface in macrophages, but still accounts for the observed specific internalization of apoA-I.