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Biomedical subjects

I Mellman

Publications and source records attributed to I Mellman.

At least 73 records · Page 4Linked to original sources
PubMed1995

Lonely MHC molecules seeking immunogenic peptides for meaningful relationships.

The association between antigenic peptides and MHC class II molecules represents a critical event in the initiation of the immune response to extracellular antigens. Understanding the molecular basis of antigen processing requires the characterization of the intracellular compartments, or 'singles bars', in which immunogenic peptides are generated and loaded onto class II molecules. In the past year, something of a breakthrough occurred with the identification of specialized compartments that host antigen processing and/or peptide loading, designated 'MHC class II compartment' and 'class II vesicles'. It is becoming increasingly clear, however, that these compartments are themselves heterogeneous and not always distinct from conventional endosomes and lysosomes.

Animals↗
PubMed1994

Rat liver endocytic coated vesicles do not exhibit ATP-dependent acidification in vitro.

Coated vesicle fractions from a variety of tissues have been found to contain a vacuolar proton ATPase. Since these fractions contain both plasma membrane- and Golgi-derived coated vesicles, we sought to determine specifically whether endocytic coated vesicles from rat liver contain an active vacuolar proton ATPase. Endocytic vesicles (coated vesicles and endosomes) were selectively labeled with pH-sensitive endocytic tracers (fluorescein isothiocyanate-dextran or -asialoorsomucoid). Coated vesicles were then separated from endosomes by sucrose density gradient centrifugation. Although the endosomal fractions were found to exhibit significant ATP-dependent acidification activity, highly purified coated vesicles containing pH-sensitive endocytic tracers were unable to generate a pH gradient in response to ATP addition. The coated vesicles could be passively acidified, however, by creating potassium diffusion potentials, indicating that they were in fact capable of maintaining proton gradients. Moreover, significant ATP-dependent acidification activity was observed when the coated vesicle fractions were assayed using the nonselective externally added pH probe acridine orange. Thus, it appears that rat liver endocytic coated vesicles do not contain a functional proton pump. The active vacuolar proton ATPase found in these fractions instead reflected the presence of Golgi-derived coated vesicles or contaminating membranes.

Adenosine Triphosphate↗
PubMed1994

Transient accumulation of new class II MHC molecules in a novel endocytic compartment in B lymphocytes.

Endocytosis of antigen by antigen-presenting cells results in the production of peptides that bind to newly synthesized class II molecules of the major histocompatibility complex. A new population of class II-enriched vesicles has been discovered in B lymphocytes that accumulate internalized antigen but are distinct from endosomes and lysosomes. These vesicles also transiently accumulate newly synthesized class II and class II-peptide complexes and appear to be a compartment specialized for the transport and loading of class II molecules.

Animals↗
PubMed1994

The invariant chain is required for intracellular transport and function of major histocompatibility complex class II molecules.

The major histocompatibility complex (MHC) class II-associated invariant chain (Ii) is thought to act as a chaperone that assists class II during folding, assembly, and transport. To define more precisely the role of Ii chain in regulating class II function, we have investigated in detail the biosynthesis, transport, and intracellular distribution of class II molecules in splenocytes from mice bearing a deletion of the Ii gene. As observed previously, the absence of Ii chain caused significant reduction in both class II-restricted antigen presentation and expression of class II molecules at the cell surface because of the intracellular accumulation of alpha and beta chains. Whereas much of the newly synthesized MHC molecules enter a high molecular weight aggregate characteristic of misfolded proteins, most of the alpha and beta chains form dimers and acquire epitopes characteristic of properly folded complexes. Although the complexes do not bind endogenously processed peptides, class II molecules that reach the surface are competent to bind peptides added to the medium, further demonstrating that at least some of the complexes fold properly. Similar to misfolded proteins, however, the alpha and beta chains are poorly terminally glycosylated, suggesting that they fail to reach the Golgi complex. As demonstrated by double label confocal and electron microscope immunocytochemistry, class II molecules were found in a subcompartment of the endoplasmic reticulum and in a population of small nonlysosomal vesicles possibly corresponding to the intermediate compartment or cis-Golgi network. Thus, although alpha and beta chains can fold and form dimers on their own, the absence of Ii chain causes them to be recognized as "misfolded" and retained in the same compartments as bona fide misfolded proteins.

Animals↗
PubMed1994

Mechanisms of cell polarity: sorting and transport in epithelial cells.

The recent discovery of widely distributed targeting determinants, which govern the polarized cell-surface distribution of plasma membrane proteins in epithelial cells, has significantly changed our view of how polarized cells generate functionally distinct membrane domains. Together with the surprising finding that the same determinants are recognized on both the biosynthetic and the endocytic pathways, it now appears likely that a common epigenetic code may exist that controls molecular sorting of membrane proteins in a wide variety of polarized, and perhaps even non-polarized, cell types.

Animals↗
PubMed1994

Dictyostelium discoideum mutants with conditional defects in phagocytosis.

We have isolated and characterized Dictyostelium discoideum mutants with conditional defects in phagocytosis. Under suspension conditions, the mutants exhibited dramatic reductions in the uptake of bacteria and polystyrene latex beads. The initial binding of these ligands was unaffected, however, indicating that the defect was not in a plasma membrane receptor: Because of the phagocytosis defect, the mutants were unable to grow when cultured in suspensions of heat-killed bacteria. The mutants exhibited normal capacities for fluid phase endocytosis and grew as rapidly as parental (AX4) cells in axenic medium. Both the defects in phagocytosis and growth on bacteria were corrected when the mutant Dictyostelium cells were cultured on solid substrates. Reversion and genetic complementation analysis suggested that the mutant phenotypes were caused by single gene defects. While the precise site of action of the mutations was not established, the mutations are likely to affect an early signaling event because the binding of bacteria to mutant cells in suspension was unable to trigger the localized polymerization of actin filaments required for ingestion; other aspects of actin function appeared normal. This class of conditional phagocytosis mutant should prove to be useful for the expression cloning of the affected gene(s).

Animals↗
PubMed1994

Structural requirements and sequence motifs for polarized sorting and endocytosis of LDL and Fc receptors in MDCK cells.

In MDCK cells, basolateral sorting of most membrane proteins has been shown to depend on distinct cytoplasmic domain determinants. These signals can be divided into those which are related to signals for localization at clathrin-coated pits and those which are unrelated. The LDL receptor bears two tyrosine-containing signals, one of each class, that can independently target receptors from the Golgi complex and from endosomes to the basolateral plasma membrane. We have now investigated the other structural features required for the activity of both determinants. We find that both depend, at least in part, on clusters of 1-3 acidic amino acids located on the COOH-terminal side of each tyrosine. While single residues adjacent to each tyrosine were also found to be critical, the two signals differed in that only the coated pit-unrelated signal could tolerate a phenylalanine in place of its tyrosine residue. We also found that the structural requirements for basolateral targeting of the "coated pit-related" signal were distinct from those required for rapid endocytosis. Apart from sharing a common tyrosine residue, no feature of the NPXY motif for coated pit localization was required for basolateral targeting. We also investigated basolateral targeting of the mouse macrophage Fc receptor (FcRII-B2) which contains a tyrosine-independent coated pit localization signal. Basolateral transport and endocytosis were found to depend on a common dileucine-type motif. Thus, basolateral targeting determinants, like coated pit domains, can contain either tyrosine- or di-leucine-containing signals. The amino acids in the vicinity of these motifs determine whether they function as determinants for endocytosis, basolateral targeting, or both.

Amino Acid Sequence↗
PubMed1994

Dictyostelium discoideum mutants with temperature-sensitive defects in endocytosis.

We have isolated and characterized temperature-sensitive endocytosis mutants in Dictyostelium discoideum. Dictyostelium is an attractive model for genetic studies of endocytosis because of its high rates of endocytosis, its reliance on endocytosis for nutrient uptake, and tractable molecular genetics. Endocytosis-defective mutants were isolated by a fluorescence-activated cell sorting (FACS) as cells unable to take up a fluorescent marker. One temperature-sensitive mutant (indy1) was characterized in detail and found to exhibit a complete block in fluid phase endocytosis at the restrictive temperature, but normal rates of endocytosis at the permissive temperature. Likewise, a potential cell surface receptor that was rapidly internalized in wild-type cells and indy1 cells at the permissive temperature was poorly internalized in indy1 under restrictive conditions. Growth was also completely arrested at the restrictive temperature. The endocytosis block was rapidly induced upon shift to the restrictive temperature and reversed upon return to normal conditions. Inhibition of endocytosis was also specific, as other membrane-trafficking events such as phagocytosis, secretion of lysosomal enzymes, and contractile vacuole function were unaffected at the restrictive temperature. Because recycling and transport to late endocytic compartments were not affected, the site of the defect's action is probably at an early step in the endocytic pathway. Additionally, indy1 cells were unable to proceed through the normal development program at the restrictive temperature. Given the tight functional and growth phenotypes, the indy1 mutant provides an opportunity to isolate genes responsible for endocytosis in Dictyostelium by complementation cloning.

Adenosine Triphosphate↗
PubMed1993

Common signals control low density lipoprotein receptor sorting in endosomes and the Golgi complex of MDCK cells.

The cytoplasmic domain of the LDL receptor bears two tyrosine-containing determinants that can independently target receptors from the Golgi to the basolateral plasma membrane of MDCK cells. We found that these determinants, localized to the membrane-proximal and -distal regions of the receptor's cytoplasmic domain, also control polarized sorting in endosomes. Inactivation of the distal determinant reduced receptors' ability to return to the basolateral domain following endocytosis, resulting instead in receptor transcytosis from basolateral endosomes to the apical plasma membrane. Similarly, receptors internalized from the apical surface were transported from apical endosomes to the basolateral surface, owing to the proximal basolateral targeting determinant. Thus, receptor recycling in endosomes is directed by the same signals as polarized sorting in the Golgi, indicating that sorting on the endocytic and biosynthetic pathways involves similar mechanisms. The observation that brefeldin A interfered with sorting but not transport in both endosomes and the Golgi further supports this.

Amino Acid Sequence↗
PubMed1993

Molecular sorting in polarized and non-polarized cells: common problems, common solutions.

We have been interested in determining how epithelial cells generate and maintain their characteristically polarized distributions of membrane proteins. Our efforts to date strongly indicate that the polarized transport in MDCK cells may be due to a set of discrete targeting determinants often found on a membrane protein's cytoplasmic domain. Surprisingly, these determinants are widely distributed and are not specific to proteins expressed in polarized cells. They also appear to function in controlling polarized transport along both the biosynthetic and the endocytic (or transcytotic) pathways. Signals for basolateral transport have been characterized and, like the cytoplasmic domain signal used by plasma membrane receptors for accumulation at clathrin-coated pits, they often involve a critical tyrosine residue. Although the basolateral and coated pit signals may also be co-linear, they are not identical. The basolateral and apical transport determinants are also hierarchically arranged. Although a single protein may contain one or more signals specifying basolateral transport, inactivation of these signals appears to reveal a determinant that directs efficient apical transport. Given that the sequence determinants responsible for polarized transport are not restricted to epithelial cells and are related to determinants commonly utilized in all cells, it is possible that non-polarized cells contain cognate apical and basolateral pathways that are responsible for 'constitutive' transport from the Golgi to the plasma membrane. The presence of two cognate pathways might confer a high degree of plasticity to pre-differentiated cells, allowing them rapidly to begin assuming a polarized phenotype in response to extracellular stimuli without requiring the synthesis of epithelial cell-specific transport machinery.

Amino Acid Sequence↗
PubMed1992

Basolateral sorting of LDL receptor in MDCK cells: the cytoplasmic domain contains two tyrosine-dependent targeting determinants.

In MDCK cells, transport of membrane proteins to the basolateral plasma membrane has been shown to require a distinct cytoplasmic domain determinant. Although the determinant is often related to signals used for localization in clathrin-coated pits, inactivation of the coated pit domain in the human LDL receptor did not affect basolateral targeting. By expressing mutant and chimeric LDL receptors, we have now identified two independently acting signals that are individually sufficient for basolateral targeting. The two determinants mediate basolateral sorting with different efficiencies, but both contain tyrosine residues critical for activity. The first determinant was colinear with, but distinct from, the coated pit domain of the receptor. The second was found in the C-terminal region of the cytoplasmic domain of the receptor and, although tyrosine-dependent, did not mediate endocytosis. The results suggest that membrane proteins can have functionally redundant signals for basolateral transport and that a tyrosine-containing motif may be a common feature of multiple intracellular sorting events.

Amino Acid Sequence↗
PubMed1992

The small GTP-binding protein rab4 controls an early sorting event on the endocytic pathway.

rab4 is a ras-like GTP-binding protein that associates with early endosomes in a cell cycle-dependent fashion. To determine its role during endocytosis, we generated stable cell lines that overexpressed mutant or wild-type rab4. By measuring endocytosis, transport to lysosomes, and recycling, we found that overexpression of wild-type rab4 had differential effects on the endocytic pathway. Although initial rates of internalization and degradation were not inhibited, the transfectants exhibited a 3-fold decrease in fluid phase endocytosis as well as an alteration in transferrin receptor (Tfn-R) recycling. Wild-type rab4 caused a redistribution of Tfn-R's from endosomes to the plasma membrane. It also blocked iron discharge by preventing the delivery of Tfn to acidic early endosomes, instead causing Tfn accumulation in a population of nonacidic vesicles and tubules. rab4 thus appears to control the function or formation of endosomes involved in recycling.

Animals↗
PubMed1992

Brefeldin A and the endocytic pathway. Possible implications for membrane traffic and sorting.

A number of recent observations have suggested that the endocytic and biosynthetic pathways may share fundamentally similar transport mechanisms at the molecular level. Some of the more striking of these suggestions have come from a comparison of the effects of the macrocyclic lactone brefeldin A (BFA) on endosomes and the Golgi complex. BFA is thought to affect Golgi-specific coat proteins that may be involved in maintaining the structural integrity of the organelle and in regulating membrane transport in the secretory pathway. Many of the effects of BFA on the endocytic system, such as the guanine nucleotide and aluminum fluoride (AlF4-)-regulated induction of microtubule-dependent endosomal tubules, are strikingly reminiscent of the action of the drug on the Golgi complex. Therefore, the similar mechanisms of action of the drug on endosomes suggest that organelles of the endocytic pathway may be associated with similar cytoplasmic coats that could regulate endosome function and integrity.

Brefeldin A↗
PubMed1992

Cytoplasmic domain heterogeneity and functions of IgG Fc receptors in B lymphocytes.

B lymphocytes and macrophages express closely related immunoglobulin G (IgG) Fc receptors (Fc gamma RII) that differ only in the structures of their cytoplasmic domains. Because of cell type-specific alternative messenger RNA splicing, B-cell Fc gamma RII contains an insertion of 47 amino acids that participates in determining receptor function in these cells. Transfection of an Fc gamma RII-negative B-cell line with complementary DNA's encoding the two splice products and various receptor mutants indicated that the insertion was responsible for preventing both Fc gamma RII-mediated endocytosis and Fc gamma RII-mediated antigen presentation. The insertion was not required for Fc gamma RII to modulate surface immunoglobulin-triggered B-cell activation. Instead, regulation of activation involved a region of the cytoplasmic domain common to both the lymphocyte and macrophage receptor isoforms. In contrast, the insertion did contribute to the formation of caps in response to receptor cross-linking, consistent with suggestions that the lymphocyte but not macrophage form of the receptor can associate with the detergent-insoluble cytoskeleton.

Amino Acid Sequence↗
PubMed1992

Reversible phosphorylation--dephosphorylation determines the localization of rab4 during the cell cycle.

The ras-like GTP binding protein rab4 is the only known rab protein on endosomes that is phosphorylated during mitosis. Since a large fraction of rab4 accumulates in the cytosol in mitotic cells, we investigated the molecular mechanism controlling membrane association of rab4. We first show that human rab4 is phosphorylated by recombinant mammalian p34cdc2 kinase in vitro. Next, the actual site of phosphorylation and its functional significance were determined using stably transfected CHO cell lines producing high levels of wild type rab4 or rab4 mutants bearing alterations at Ser196, which occurs within a consensus site for p34cdc2 kinase phosphorylation (S196PRR). Mutation of Ser196 to glutamine or aspartic acid completely prevented rab4 phosphorylation in mitotic cells and also blocked its appearance in the cytosol. Neither C-terminal isoprenylation nor carboxymethylation of rab4 was affected by the mutations or by phosphorylation. Finally, dephosphorylation and reassociation of soluble rab4 with membranes occurred upon exit of cells from mitosis. Thus, phosphorylation of Ser196 is directly responsible for the reversible translocation of rab4 into the cytosol of mitotic cells.

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