Reconstitution of endocytosis and recycling using perforated Madin-Darby canine kidney cells.
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Animals↗
Biomedical subjects
I Mellman
Publications and source records attributed to I Mellman.
Fc receptor endocytosis is controlled by a cytoplasmic domain determinant that actively prevents coated pit localization.
Macrophages and B-lymphocytes express two major isoforms of Fc receptor (FcRII-B2 and FcRII-B1) that exhibit distinct capacities for endocytosis. This difference in function reflects the presence of an in-frame insertion of 47 amino acids in the cytoplasmic domain of the lymphocyte isoform (FcRII-B1) due to alternative mRNA splicing. By expressing wild type and mutant FcRII cDNAs in fibroblasts, we have now examined the mechanism by which the insertion acts to prevent coated pit localization and endocytosis. We first identified the region of the FcRII-B2 cytoplasmic domain that is required for rapid internalization. Using a biochemical assay for endocytosis and an immuno-EM assay to determine coated pit localization directly, we found that the distal half of the cytoplasmic domain, particularly a region including residues 18-31, as needed for coated pit-mediated endocytosis. Elimination of the tyrosine residues at position 26 and 43, separately or together, had little effect on coated pit localization and a partial effect on endocytosis of ligand. Since the FcRII-B1 insertion occurs in the membrane-proximal region of the cytoplasmic domain (residue 6) not required for internalization, it is unlikely to act by physically disrupting the coated pit localization determinant. In fact, the insertion was found to prevent endocytosis irrespective of its position in the cytoplasmic tail and appeared to selectively exclude the receptor from coated regions. Moreover, receptors bearing the insertion exhibited a temperature- and ligand-dependent association with a detergent-insoluble fraction and with actin filaments, perhaps in part explaining the inability of FcRII-B1 to enter coated pits.
Actins↗
Transport of the lysosomal membrane glycoprotein lgp120 (lgp-A) to lysosomes does not require appearance on the plasma membrane.
We have used stably transfected CHO cell lines to characterize the pathway of intracellular transport of the lgp120 (lgp-A) to lysosomes. Using several surface labeling and internalization assays, our results suggest that lgp120 can reach its final destination with or without prior appearance on the plasma membrane. The extent to which lgp120 was transported via the cell surface was determined by two factors: expression level and the presence of a conserved glycine-tyrosine motif in the cytoplasmic tail. In cells expressing low levels of wild-type lgp120, the majority of newly synthesized molecules reached lysosomes without becoming accessible to antibody or biotinylation reagents added extracellularly at 4 degrees C. With increased expression levels, however, an increased fraction of transfected lgp120, as well as some endogenous lgp-B, appeared on the plasma membrane. The fraction of newly synthesized lgp120 reaching the cell surface was also increased by mutations affecting the cytoplasmic domain tyrosine or glycine residues. A substantial fraction of both mutants reached the surface even at low expression levels. However, only the lgp120G----A7 mutant was rapidly internalized and delivered from the plasma membrane to lysosomes. Taken together, our results show that the majority of newly synthesized wild-type lgp120 does not appear to pass through the cell surface en route to lysosomes. Instead, it is likely that lysosomal targeting involves a saturable intracellular sorting site whose affinity for lgp's is dependent on a glycine-tyrosine motif in the lgp120 cytoplasmic tail.
Animals↗
The importance of being acid: the role of acidification in intracellular membrane traffic.
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Adenosine Triphosphatases↗
Selective inhibition of transcytosis by brefeldin A in MDCK cells.
Treatment of most cells with brefeldin A (BFA) leads to the retrieval of the Golgi complex to the endoplasmic reticulum, presumably reflecting an inhibition of cytoplasmic coat protein binding to Golgi membranes. Although BFA has been thought to act only on biosynthetic organelles, we now show that this drug also reversibly blocks polymeric immunoglobulin receptor-mediated transcytosis in MDCK cells. The action of BFA on transcytosis was selective, since internalization, recycling, and intracellular degradation were unaffected. The block occurred early on the transcytotic pathway, probably before the translocation of IgA-containing vesicles from the basal to the apical cytoplasm. Although BFA caused MDCK cell endosomes to become more tubular, the organization of the Golgi and binding of the 110 kd Golgi coat protein beta-COP was surprisingly unaffected. These results suggest that in MDCK cells, endocytic organelles contain a BFA-sensitive coat that regulates their organization and function even though the Golgi coat is BFA resistant.
Animals↗
Accumulation of indigestible substances reduces fusion competence of macrophage lysosomes.
It is well known that mouse macrophages loaded with indigestible substances become highly vacuolated. However, why this vacuolization occurs and its effect on lysosome function and intracellular transport during endocytosis remain unknown. Here, macrophage vacuoles were formed by incubation with sucrose or a tripeptide of the D-isomer of alanine and were determined to be lysosomal in origin by staining with the lysosomal glycoproteins and lysosomal hydrolases. However, as indicated by confocal and electron microscopy, subsequent delivery of both fluid phase (lucifer yellow, horse-radish peroxidase) and receptor-bound ligands (IgG complexes) was significantly reduced, suggesting that indigestible material reduced the ability of the loaded lysosomes to fuse with endosomes containing newly internalized tracers. Nevertheless, ligands internalized by the vacuolated cells were degraded at almost the normal rate, indicating that degradation occurs in the absence of delivery to the loaded lysosomes. We have also found that this fusion inhibition occurs in human alveolar macrophages loaded with physiologic debris from smoking and asbestos. These results suggest that indigestible material within lysosomes, such as is present in residual bodies in vivo, may affect their fusion competence.
Animals↗
Basolateral sorting in MDCK cells requires a distinct cytoplasmic domain determinant.
In MDCK cells, Golgi to basolateral transport of several membrane proteins has been found to involve a cytoplasmic domain determinant. In some cases (Fc receptor, lysosomal glycoprotein Igp120), the determinant appears similar to that required for endocytosis via clathrin-coated pits; for Igp120, elimination of a single cytoplasmic domain tyrosine both blocks internalization and results in apical transport. In other cases (LDL receptor), the determinant does not involve the cytoplasmic domain tyrosine required for endocytosis. Thus, contrary to current models, basolateral transport in MCDK cells occurs not by default but depends on one or more cytoplasmic domain determinants, the precise nature of which is unknown. For some proteins, it is closely related to coated pit determinants. The fact that many membrane proteins can reach the apical surface in the absence of this determinant suggests that signals for apical transport are widely distributed.
Amino Acid Sequence↗
The small GTP-binding protein rab4 is associated with early endosomes.
Small GTP-binding proteins of the rab family have been implicated as playing important roles in controlling membrane traffic on the biosynthetic and endocytic pathways. We demonstrate that a distinct rab protein, rab4p, is associated with the population of early endosomes involved in transferrin-receptor recycling. An antibody to human rab4p was found to detect a doublet of approximately 24-kDa proteins on immunoblots from various cell types. Seventy-five percent of these proteins were tightly membrane bound and could be released only by detergent treatment. Upon isolation of early endosomes, late endosomes, and lysosomes, by free-flow electrophoresis and Percoll density-gradient centrifugation, most (70%) of the rab4p was found to cofractionate with early endosomes and endocytic vesicles containing 125I-labeled transferrin. The rab proteins previously localized to the endoplasmic reticulum and/or Golgi apparatus were not found in these fractions. We also localized rab4p to transferrin-receptor-containing early endosomes by immunofluorescence after expression of rab4 cDNA. The association of rab4p with early endosomes and other vesicles involved in the intracellular transport of transferrin receptor suggests that rab4p may play a role in regulating the pathway of receptor recycling.
Animals↗
Relationships between sorting in the exocytic and endocytic pathways of MDCK cells.
Epithelial cells use both the exocytic and endocytic pathways to generate and maintain the polarized distribution of membrane proteins. This review summarizes current information concerning the general features and functions of the exocytic and endocytic pathways in MDCK cells. We analyse the possible implications of similarities between signals for endocytosis and determinants for basolateral sorting in the TGN. Furthermore, we discuss the fundamental relationships that might also exist in the biochemical basis of membrane traffic in the two pathways.
Brefeldin A↗
Toxoplasma gondii: fusion competence of parasitophorous vacuoles in Fc receptor-transfected fibroblasts.
After actively entering its host cells, the protozoan parasite Toxoplasma gondii resides in an intracellular vacuole that is completely unable to fuse with other endocytic or biosynthetic organelles. The fusion blocking requires entry of viable organisms but is irreversible: fusion competence of the vacuole is not restored if the parasite is killed after entry. The fusion block can be overcome, however, by altering the parasite's route of entry. Thus, phagocytosis of viable antibody-coated T. gondii by Chinese hamster ovary cells transfected with macrophage-lymphocyte Fc receptors results in the formation of vacuoles that are capable of both fusion and acidification. Phagocytosis and fusion appear to involve a domain of the Fc receptor cytoplasmic tail distinct from that required for localization at clathrin-coated pits. These results suggest that the mechanism of fusion inhibition is likely to reflect a modification of the vacuole membrane at the time of its formation, as opposed to the secretion of a soluble inhibitor by the parasite.
Animals↗
Characterization and cloning of lgp110, a lysosomal membrane glycoprotein from mouse and rat cells.
lgp110 is a heavily glycosylated intrinsic protein of lysosomal membranes. Initially defined by monoclonal antibodies against mouse liver lysosomes, it consists of a 45-kilodalton core polypeptide with O-linked and 17 asparagine-linked oligosaccharide side chains in mouse cells. Sialic acid residues make the mature protein extremely acidic, with an isoelectric point of between 2 and 4 in both normal tissues and most cultured cell lines. Partial sequencing of mouse lgp110 allowed oligonucleotide probes to be constructed for the screening of several mouse cDNA libraries. A partial cDNA clone for mouse lgp110 was found and used for additional library screening, generating a cDNA clone covering all of the coding sequence of mature rat lgp110 as well as genomic clones covering most of the mouse gene. These new clones bring to seven the number of lysosomal membrane proteins whose amino acid sequences can be deduced, and two distinct but highly similar groups (designated lgp-A and lgp-B) can now be defined. Sequence comparisons suggest that differences within each group reflect species variations of the same protein and that lgp-A and lgp-B probably diverged from a common ancestor prior to the evolup4f1ary divergence of birds and mammals. Individual cells and individual lysosomes possess both lgp-A and lgp-B, suggesting that these two proteins have different functions. Mouse lgp110 is encoded by at least seven exons; intron positions suggest that the two homologous ectodomains of each lgp arose through gene duplication.
Amino Acid Sequence↗
Fc receptor phosphorylation during receptor-mediated control of B-cell activation.
It is well known that Fc receptors for IgG (FcRII) on macrophages mediate the endocytosis of antibody-antigen complexes and signal the release of inflammatory and cytotoxic agents. FcRII are also expressed at high levels on B cells where they are less involved in endocytosis than in modulating B-cell activation by membrane immunoglobulins. Although crosslinking of membrane immunoglobulins can result in B-cell differentiation and proliferation through stimulation of phospholipase C, mobilization of intracellular Ca2+, and activation of protein kinase C, crosslinking FcR with membrane immunoglobulins confers a dominant inhibitory signal that prevents or aborts activation. This form of regulation may have a role in the induction of tolerance by IgG and in controlling the B-cell repertoire by anti-idiotypes. The different functions of FcR on B cells and macrophages may reflect the fact that these cell types express closely related but distinct FcR isoforms. We have recently found that the main lymphocyte FcR isoform, FcRII-B1, is unable to mediate endocytosis by way of coated pits and coated vesicles owing to an in-frame insertion of 47 amino acids in its cytoplasmic tail. Here we show that this insert, absent from the FcRII-B2 macrophage isoform, also contains serine phosphorylation sites that may have a role in the ability of FcR to regulate B-cell activation through membrane immunoglobulins.
Alkaloids↗
ATP and cytosol requirements for transferrin recycling in intact and disrupted MDCK cells.
We have developed an in vitro system for studying membrane transport during receptor-mediated endocytosis. Using nitrocellulose disruption to permeabilize selectively the apical domain of filter-grown MDCK cells, the recycling of receptor-bound transferrin (Tfn) from an intracellular pool was reconstituted in vitro with a rate and efficiency similar to that of intact cells. Tfn and Tfn receptor recycling from endosomes back to the cell surface was dependent on added ATP and cytosol-derived proteins. Thus, incubation of intact cells under conditions of ATP depletion resulted in the clearance of Tfn receptors from the basolateral membrane, this was reversible upon removal of the energy poisons. Reappearance of previously internalized receptors could also be obtained in disrupted cells but required the addition of both ATP and cytosol to the assay mixture. Similarly, when intact cells were allowed to internalize labeled Tfn prior to disruption, efficient and rapid release of ligand back into the medium was markedly stimulated by ATP and cytosol. Recycling was judged to be both selective and vectorial since only the expected small fraction of a previously internalized horseradish peroxidase was released after addition of ATP and cytosol, and release was primarily into the basal medium. While the cytosol contributed one or more protein factors, none was sensitive to N-ethylmaleimide. Alkylation of the disrupted cells, however, did inactivate recycling.
Adenosine Triphosphate↗
Differential microtubule requirements for transcytosis in MDCK cells.
Given the role of microtubules in directing the transport of many intracellular organelles, we investigated whether intact microtubules were also required for transcytosis across epithelia. Using polarized MDCK cells expressing receptors for the Fc domain of IgG (FcRII-B2) or polymeric immunoglobulin (pIg-R), we examined the involvement of microtubules in apical to basolateral and basolateral to apical transcytosis, respectively. While depolymerization of microtubules with nocodozole had no effect on apical to basolateral transcytosis via FcR, basolateral to apical transcytosis of dimeric IgA via pIg-R was almost completely blocked. Inhibition due to nocodozole was selective for basolateral to apical transcytosis, since neither endocytosis nor receptor recycling was significantly affected at either plasma membrane domain. As shown by confocal microscopy, the block in transcytosis was due to the inability of MDCK cells to translocate IgA-containing vesicles from the basolateral to the apical cytoplasm in the absence of an intact microtubule network. The nocodazole sensitive step could be partially by-passed, however, by allowing cells to internalize IgA at 17 degrees C prior to nocodazole treatment. Although incubation at 17 degrees C blocked release of IgA into the apical medium, it did not prevent translocation of IgA-containing vesicles to the apical cytoplasm. Thus, receptor-mediated transcytosis in opposite directions exhibits distinct requirements for microtubules, a feature which reflects the spatial organization of MDCK cells.
Animals↗
Endocytosis and antigen processing.
It has become increasingly clear that endocytosis plays a critical role in the processing of exogenous antigens for presentation to T lymphocytes. However, many critical questions remain concerning the pathways taken by antigens and MHC molecules following internalization by antigen presenting cells. This review summarizes current information concerning the general features and functions of the endocytic pathway in mammalian cells. We also identify the likely roles of the various endocytic organelles in each of the events associated with antigen processing.
Animals↗
Human IgG Fc receptor (hFcRII; CD32) exists as multiple isoforms in macrophages, lymphocytes and IgG-transporting placental epithelium.
We previously isolated cDNA clones from a human monocyte library that encoded one member of a family of low-affinity surface receptors for the Fc domain of IgG (hFcRII-A). To investigate possible structural and functional heterogeneity among these receptors, we have now isolated two additional cDNAs (hFcRII-B and hFcRII-C) from a human placental library, placenta being a good source of FcR-bearing macrophages and epithelial cells. Three cDNAs encoded related but distinct transmembrane glycoproteins containing two immunoglobulin-like domains; however, transfected cells produced receptors that were indistinguishable on the basis of ligand binding or reactivity with anti-hFcRII monoclonal antibodies. The sequences of hFcRII-A and -B were most closely related and were identical except for several amino acid substitutions and one small internal deletion. While the ectodomain of hFcRII-C was identical to hFcRII-B, its cytoplasmic tail was unrelated but highly homologous to the corresponding domain of the receptor isoform (mFcRII-B2) found in murine macrophages. Thus, human FcRII may be derived from at least two alternatively spliced genes. Northern blots revealed little difference in the pattern of expression of hFcRII isoforms among various myeloid and lymphoid cells or cell lines. However, the blots--as well as in situ hybridization and immunohistochemistry--demonstrated that hFcRII-C (along with a second monocyte marker, the c-fms encoded CSF-1 receptor) was expressed in placental syncytiotrophoblasts. Since syncytiotrophoblasts comprise the IgG-transporting epithelium of the placental villus, these findings suggest that FcR found in the immune system and in certain epithelia may be structurally or functionally related.
Amino Acid Sequence↗
Fc receptor isoforms exhibit distinct abilities for coated pit localization as a result of cytoplasmic domain heterogeneity.
Mouse macrophages and lymphocytes express two distinct isoforms of a single class of Fc receptor for IgG. The macrophage isoform (FcRII-B2) is identical to the lymphocyte isoform (FcRII-B1) except for an inframe insertion in the cytoplasmic tail of FcRII-B1 that increases its length from 47 to 94 amino acids. To determine the functional significance of this cytoplasmic domain variation, presumably the result of alternative mRNA splicing, we expressed both isoforms in receptor-negative fibroblasts. While FcRII-B2 mediated the efficient ligand internalization and delivery to lysosomes, endocytosis via FcRII-B1--and via a tailminus mutant--was relatively inefficient. This difference reflected the inability of FcRII-B1 (and the tailminus mutant) to accumulate in clathrin-coated pits. Thus, the FcRII-B2 cytoplasmic tail contains a domain needed for accumulation in coated pits, and this domain is disrupted by the 47 amino acid insertion in FcRII-B1.
Animals↗
Acidification and ion permeabilities of highly purified rat liver endosomes.
While it is well established that acidic pH in endosomes plays a critical role in mediating the orderly traffic of receptors and ligands during endocytosis, little is known about the bioenergetics or regulation of endosome acidification. Using highly enriched fractions of rat liver endosomes prepared by free flow electrophoresis and sucrose density gradient centrifugation, we have analyzed the mechanism of ATP-dependent acidification and ion permeability properties of the endosomal membrane. This procedure permitted the isolation of endosome fractions which were up to 200-fold enriched as indicated by the increased specific activity of ATP-dependent proton transport. Acidification was monitored using hepatocyte and total liver endosomes selectively labeled with pH-sensitive markers of receptor-mediated endocytosis (fluorescein isothiocyanate asialoorosomucoid) or fluid-phase endocytosis (fluorescein isothiocyanate-dextran). In addition, changes in membrane potential accompanying ATP-dependent acidification were directly measured using the voltage-sensitive fluorescent dye Di-S-C3(5). Our results indicate that ATP-dependent acidification of liver endosomes is electrogenic, with proton transport being accompanied by the generation of an interior-positive membrane potential opposing further acidification. The membrane potential can be dissipated by the influx of permeant external anions or efflux of internal alkali cations. Replacement externally of permeable anions with less permeable anions (e.g. replacing Cl- with gluconate) diminished acidification, as did replacement internally of a more permeant cation K+ with less permeant species (such as Na+ or tetramethylammonium). ATP-dependent H+ transport was not coupled to any specific anion or cation, however. The endosomal membrane was found to be extremely permeable to protons, with protons able to leak out almost as fast as they are pumped in. Thus, the internal pH of endosomes is likely to reflect a dynamic equilibrium of protons regulated by the intrinsic ion permeabilities of the endosomal membrane, in addition to the activity of an ATP-driven proton pump.
Animals↗