p110-related PI 3-kinases regulate phagosome-phagosome fusion and phagosomal pH through a PKB/Akt dependent pathway in Dictyostelium.

The Dictyostelium p110-related PI 3-kinases, PIK1 and PIK2, regulate the endosomal pathway and the actin cytoskeleton, but do not significantly regulate internalization of particles in D. discoideum. Bacteria internalized into delta ddpik1/ddpik2 cells or cells treated with PI 3-kinase inhibitors remained intact as single particles in phagosomes with closely associated membranes after 2 hours of internalization, while in control cells, bacteria appeared degraded in multi-particle spacious phagosomes. Addition of LY294002 to control cells, after 60 minutes of chase, blocked formation of spacious phagosomes, suggesting PI 3-kinases acted late to regulate spacious phagosome formation. Phagosomes purified from control and drug treated cells contained equivalent levels of lysosomal proteins, including the proton pump complex, and were acidic, but in drug treated cells and delta ddpik1/ddpik2 cells phagosomal pH was significantly more acidic during maturation than the pH of control phagosomes. Inhibition of phagosomal maturation by LY294002 was overcome by increasing phagosomal pH with NH(4)Cl, suggesting that an increase in pH might trigger homotypic phagosome fusion. A pkbA null cell line (PKB/Akt) reproduced the phenotype described for cells treated with PI 3-kinase inhibitors and delta ddpik1/ddpik2 cells. We propose that PI 3-kinases, through a PKB/Akt dependent pathway, directly regulate homotypic fusion of single particle containing phagosomes to form multi-particle, spacious phagosomes, possibly through the regulation of phagosomal pH.

Dietary iron induces rapid changes in rat intestinal divalent metal transporter expression.

The divalent metal transporter (DMT1, also known as NRAMP2 or DCT1) is the likely target for regulation of intestinal iron absorption by iron stores. We investigated changes in intestinal DMT1 expression after a bolus of dietary iron in iron-deficient Belgrade rats homozygous for the DMT1 G185R mutation (b/b) and phenotypically normal heterozygous littermates (+/b). Immunofluorescent staining with anti-DMT1 antisera showed that DMT1 was located in the brush-border membrane. Duodenal DMT1 mRNA and protein levels were six- and twofold higher, respectively, in b/b rats than in +/b rats. At 1.5 h after dietary iron intake in +/b and b/b rats, DMT1 was internalized into cytoplasmic vesicles. At 1.5 and 3 h after iron intake in +/b and b/b rats, there was a rapid decrease of DMT1 mRNA and a transient increase of DMT1 protein. The decrease of DMT1 mRNA was specific, because ferritin mRNA was unchanged. After iron intake, an increase in ferritin protein and decrease in iron-regulatory protein binding activity occurred, reflecting elevated intracellular iron pools. Thus intestinal DMT1 rapidly responds to dietary iron in both +/b and b/b rats. The internalization of DMT1 may be an acute regulatory mechanism to limit iron uptake. In addition, the results suggest that in the Belgrade rat DMT1 with the G185R mutation is not an absolute block to iron.

Dictyostelium lysosomal proteins with different sugar modifications sort to functionally distinct compartments.

Many Dictyostelium lysosomal enzymes contain mannose-6-phosphate (Man-6-P) in their N-linked oligosaccharide chains. We have now characterized a new group of lysosomal proteins that contain N-acetylglucosamine-1-phosphate (GlcNAc-1-P) linked to serine residues. GlcNAc-1-P-containing proteins, which include papain-like cysteine proteinases, cofractionate with the lysosomal markers and are in functional vesicles of the endosomal/lysosomal pathway. Immunoblots probed with reagents specific for each carbohydrate modification indicate that the lysosomal proteins are modified either by Man-6-P or GlcNAc-1-P, but not by both. Confocal microscopy shows that the two sets of proteins reside in physically and functionally distinct compartments. Vesicles with GlcNAc-1-P fuse with nascent bacteria-loaded phagosomes less than 3 minutes after ingestion, while those with Man-6-P do not participate in bacterial digestion until about 15 minutes after phagocytosis. Even though both types of vesicles fuse with phagosomes, GlcNAc-1-P- and Man-6-P-bearing proteins rarely colocalize. Since both lysosomal enzymes and their bound carbohydrate modifications are stable in lysosomes, a targeting or retrieval mechanism based on these carbohydrate modifications probably establishes and/or maintains segregation.

Evidence for a recycling role for Rab7 in regulating a late step in endocytosis and in retention of lysosomal enzymes in Dictyostelium discoideum.

The mammalian small molecular weight GTPase Rab7 (Ypt7 in yeast) has been implicated in regulating membrane traffic at postinternalization steps along the endosomal pathway. A cDNA encoding a protein 85% identical at the amino acid level to mammalian Rab7 has been cloned from Dictyostelium discoideum. Subcellular fractionation and immunofluorescence microscopy indicated that Rab7 was enriched in lysosomes, postlysosomes, and maturing phagosomes. Cell lines were generated that overexposed Rab7 wild-type (WT), Rab7 Q67L (constitutively active form), and Rab7 T22N (dominant negative form) proteins. The Rab7 T22N cell line internalized fluid phase markers and latex beads (phagocytosis) at one-third the rate of control cells, whereas Rab7 WT and Rab7 Q67L cell lines were normal in uptake rates but exocytosed fluid phase faster than control cells. In contrast, fluid phase markers resided in acidic compartments for longer periods of time and were more slowly exocytosed from Rab7 T22N cells as compared with control cells. Light microscopy indicated that Rab7-expressing cell lines contained morphologically altered endosomal compartments. Compared with control cells, Rab7 WT- and Rab7 Q67L-expressing cells contained a reduced number of vesicles, the size of postlysosomes (> 2.5 microns) and an increased number of smaller vesicles, many of which were nonacidic; in control cells, > 90% of the smaller vesicles were acidic. In contrast, Rab7 T22N cells contained an increased proportion of large acidic vesicles relative to nonacidic vesicles. Radiolabel pulse-chase experiments indicated that all of the cell lines processed and targeted lysosomal alpha-mannosidase normally, indicating the lack of a significant role for Rab7 in the targeting pathway; however, retention of mature lysosomal hydrolases was affected in Rab7 WT and Rab7 T22N cell lines. Contrary to the results observed for the fluid phase efflux experiments, Rab7 T22N cells oversecreted alpha-mannosidase, whereas Rab7 WT cells retained this hydrolase as compared with control cells. These data support a model that Rab7 may regulate retrograde transport of lysosomal enzymes and the V-type H(+)-ATPase from postlysosomes to lysosomes coupled with the efficient release of fluid phase from cells.

Phagosomal proteins of Dictyostelium discoideum.

In recognizing food particles. Dictyostelium cell-surface molecules initiate cytoskeletal rearrangements that result in phagosome formation. After feeding D. discoideum cells latex beads, early phagosomes were isolated on sucrose step gradients. Protein analyses of these vesicles showed that they contained glycoproteins and surface-labeled species corresponding to integral plasma membrane proteins. Cytoskeletal proteins also were associated with phagosomes, including myosin II, actin and a 30 kDa-actin bundling protein. As seen by the acridine orange fluorescence of vesicles containing bacteria, phagosomes were acidified rapidly by a vacuolar H(+)-ATPase that was detected by immunoblotting. Except for the loss of cytoskeletal proteins, few other changes over time were noted in the protein profiles of phagosomes, suggesting that phagosome maturation was incomplete. The indigestibility of the beads possibly inhibited further endocytic processing, which has been observed by others. Since nascent phagosomes contained molecules of both the cytoskeleton and plasma membrane, they will be useful in studies aimed at identifying specific protein associations occurring between membrane proteins and the cytoskeleton during phagocytosis.

Late events in the intracellular sorting of major histocompatibility complex class II molecules are regulated by the 80-82 segment of the class II beta chain.

The molecular mechanisms that regulate sorting of major histocompatibility complex (MHC) class II molecules into the endocytic pathway are poorly understood. For many proteins, access to endosomal compartments is regulated by cytosolically expressed sequences. We present evidence that a sequence in the lumenal domain of the MHC class II molecule regulates a very late event in class II biogenesis. Class II molecules containing single amino acid changes in the highly conserved 80-82 region of the beta chain were introduced into invariant chain (Ii)-negative fibroblasts with wild-type alpha chain, and the derived transfectants were analyzed biochemically. Using an endosomal isolation technique, we have quantified the level of class II molecules expressed in endocytic compartments and found that in the absence of Ii, approximately 15% of total cellular class II molecules can be isolated from endosomal compartments. Mutation at position 80 enhances this localization, while changes at positions 81 and 82 ablate class II expression in endosomal compartments. In addition, we have evaluated whether the induced changes in intracellular distribution of class II molecules were due to alterations in early biosynthetic events, indicative of misfolding of the molecules, or to modulation of later trafficking events more likely to be a consequence of the modulation of a specific transport event. Despite the dramatic effects on endosomal localization induced by the mutations, early biosynthetic events and maturation of class II were unaffected by the mutations. Collectively, our data argue that late trafficking events that control the ability of the class II molecule to access antigens is regulated by the 80-82 segment of the MHC class II beta chains.

A role for a Rab4-like GTPase in endocytosis and in regulation of contractile vacuole structure and function in Dictyostelium discoideum.

The small Mr Rab4-like GTPase, RabD, localizes to the endosomal pathway and the contractile vacuole membrane system in Dictyostelium discoideum. Stably transformed cell lines overexpressing a dominant negative functioning RabD internalized fluid phase marker at 50% of the rate of wild-type cells. Mutant cells were also slower at recycling internalized fluid. Microscopic and biochemical approaches indicated that the transport of fluid to large postlysosome vacuoles was delayed in mutant cells, resulting in an accumulation in acidic smaller vesicles, probably lysosomes. Also, RabD N121I-expressing cell lines missorted a small but significant percentage of newly synthesized lysosomal alpha-mannosidase precursor polypeptides. However, the majority of the newly synthesized alpha-mannosidase was transported with normal kinetics and correctly delivered to lysosomes. Subcellular fractionation and immunofluorescent microscopy indicated that in mutant cells contractile vacuole membrane proteins were associated with compartments morphologically distinct from the normal reticular network. Osmotic tests revealed that the contractile vacuole functioned inefficiently in mutant cells. Our results suggest that RabD regulates membrane traffic along the endosomal pathway, and that this GTPase may play a role in regulating the structure and function of the contractile vacuole system by facilitating communication with the endosomal pathway.

Involvement of the vacuolar proton-translocating ATPase in multiple steps of the endo-lysosomal system and in the contractile vacuole system of Dictyostelium discoideum.

We have investigated the effects of Concanamycin A (CMA), a specific inhibitor of vacuolar type H(+)-ATPases, on acidification and function of the endo-lysosomal and contractile vacuole (CV) systems of D. discoideum. This drug inhibited acidification and increased the pH of endo-lysosomal vesicles both in vivo and in vitro in a dose dependent manner. Treatment also inhibited endocytosis and exocytosis of fluid phase, and phagocytosis of latex beads. This report also confirms our previous conclusions (Cardelli et al. (1989) J. Biol. Chem. 264, 3454-3463) that maintenance of acidic pH in lumenal compartments is required for efficient processing and targeting of a lysosomal enzyme, alpha-mannosidase. CMA treatment compromised the function of the contractile vacuole complex as amoebae exposed to a hypo-osmotic environment in the presence of CMA, swelled rapidly and ruptured. Fluorescence microscopy revealed that CMA treatment induced gross morphological changes in D. discoideum cells, characterized by the formation of large intracellular vacuoles containing fluid phase. The reticular membranes of the CV system were also no longer as apparent in drug treated cells. Finally, this is the first report describing cells that can adapt in the presence of CMA; in nutrient medium, D. discoideum overcame the effects of CMA after one hour of drug treatment even in the absence of protein synthesis. Upon adaptation to CMA, normal sized endo-lysosomal vesicles reappeared, endo-lysosomal pH decreased, and the rate of endocytosis, exocytosis and phagocytosis returned to normal. This study demonstrates that the V-H(+)-ATPase plays an important role in maintaining the integrity and function of the endo-lysosomal and CV systems and that D. discoideum can compensate for the loss of a functional V-H(+)-ATPase.

Analysis of successive endocytic compartments isolated from Dictyostelium discoideum by magnetic fractionation.

A colloidal iron probe was fed to the amoeba Dictyostelium discoideum and chased for different intervals. Successive segments of the endocytic pathway were then isolated magnetically at high yield and purity. There were approx. 500 endocytic vacuoles per cell; their diameters increased from approx. 0.1-0.2 microns after 3 min of feeding to approx. 2 microns after 15 min of feeding and 60 min of chase. The wave-like progression of ingested probes along the endocytic pathway suggested that the transfer of cargo involved a maturation mechanism rather than the shuttling of cargo between stable compartments. The lifetime of primary pinosomes was calculated to be approx. 1 s. Multivesicular bodies were common in the 3 min fraction and abundant in 15 min lysosomes. alpha- and beta-adaptins of molecular masses of approx. 89 and 83 kDa were richer in the 3 min vesicles than in plasma membranes and later endocytic vacuoles. Acid phosphatase, intrinsic vacuole acidity, the vacuolar proton pump protein and pump activity were present at all endocytic stages but rose between the 3 min and 15 min vacuoles and declined thereafter. Bis(monoacyglycero)phosphate or BMP, a lipid characteristic of lysosomes, followed a similar time course; it contributed up to half of the total lipid in lysosomal vacuoles. We conclude that there is both continuity and differentiation along this endocytic pathway.

A Rab4-like GTPase in Dictyostelium discoideum colocalizes with V-H(+)-ATPases in reticular membranes of the contractile vacuole complex and in lysosomes.

In the course of screening a cDNA library for ras-related Dictyostelium discoideum genes, we cloned a 0.7 kb cDNA (rabD) encoding a putative protein that was 70% identical at the amino acid level to human Rab4. Rab4 is a small M(r) GTPase, which belongs to the Ras superfamily and functions to regulate endocytosis in mammalian cells. Southern blot analysis indicated that the rabD cDNA was encoded by a single copy gene while Northern blot analysis revealed that the rabD gene was expressed at relatively constant levels during growth and differentiation. Affinity-purified antibodies were prepared against a RabD fusion protein expressed in bacteria; the antibodies recognized a single 23 kDa polypeptide on western blots of cell extracts. Density gradient fractionation revealed that the RabD antigen co-distributed primarily with buoyant membranes rich in vacuolar protons pumps (V-H(+)-ATPases) and, to a lesser extent, with lysosomes. This result was confirmed by examining cell lines expressing an epitope-tagged version of RabD. Magnetically purified early endocytic vesicles and post-lysosomal vacuoles reacted more weakly with anti-RabD antibodies than did lysosomes. Other organelles were negative for RabD. Double-label indirect immunofluorescence microscopy revealed that RabD and the 100 kDa V-H(+)-ATPase subunit colocalized in a fine reticular network throughout the cytoplasm. This network was reminiscent of spongiomes, the tubular elements of the contractile vacuole system. Immunoelectron microscopy confirmed the presence of RabD in lysosome fractions and in the membranes rich in V-H(+)-ATPase. We conclude that a Rab4-like GTPase in D. discoideum is principally associated with the spongiomes of contractile vacuole complex.

Characterization of lysosomal membrane proteins of Dictyostelium discoideum. A complex population of acidic integral membrane glycoproteins, Rab GTP-binding proteins and vacuolar ATPase subunits.

Highly purified lysosomes, prepared by magnetic fractionation of homogenates from Dictyostelium discoideum cells fed colloidal iron, were lysed under hypoosmotic conditions, and the membrane-associated proteins were subjected to gel electrophoresis. Thirteen major membrane polypeptides, ranging in molecular weight from 25,000 to 100,000 were identified. The isoelectric points of these proteins ranged from below 3.8 to greater than 7.0. Most of these proteins were stripped from membranes exposed to a chaotropic agent, 3,5-diodo-2-hydroxybenzoic acid lithium salt, and were therefore classified as peripheral membrane proteins. Twenty five glycoprotein species were detected by lectin blot analysis; 19 were classified as integral membrane proteins, and were, in general, larger than 45 kDa and negatively charged due in part to the presence of mannose 6-sulfate. Western blot analysis also demonstrated that a Rab 4-like GTPase, a Rab 7-like GTPase, and at least three subunits of the vacuolar ATPase were associated with the lysosomal membrane; the ATPase subunits appeared to be major proteins in lysosomal membranes. Finally, based on N-terminal sequence analysis of a major 41-kDa lysosome-associated membrane protein, we cloned a cDNA that encodes a protein (DVA41) highly homologous to a yeast and a bovine vacuolar ATPase subunit of approximately 41 kDa. The D. discoideum DVA41 gene was apparently a single copy gene, expressed at constant levels during growth and development.

Characterization of lysosomes isolated from Dictyostelium discoideum by magnetic fractionation.

Superparamagnetic particles were prepared with iron oxide cores of congruent to 8 nm diameter and dextran coats. After feeding the probe to the amoeba, Dictyostelium discoideum, for 15 min and chasing for 15 min, a lysosome fraction was isolated magnetically. Isolates contained 76% of ingested iron, 82% of ingested fluorescent dextran, 1.3% of cell protein, 4% of the lipid, 28% of acid phosphatase, and 5% of the vacuolar H(+)-ATPase. Enrichment in endocytic markers was congruent to 60-fold; markers for other organelles were < 0.5%. The lysosomes were homogeneous, round (0.4-1.1 microns in diameter), and frequently adherent to one another through zones of intimate apposition. Cells were also fed the iron probe continuously for 3 h to fill their entire endocytic pathway; in this case, isolates contained 3.3% of cell protein, 11% of lipid, and 49% of cell acid phosphatase. Bis(monoacylglycerol)phosphate (BMP), a lipid characteristic of lysosomes in animal cells, comprised congruent to 6% of biosynthetically labelled cell lipids and up to half of the lipid in the endocytic pathway. Essentially all of the cellular BMP was recovered in isolates prepared after 3 h of feeding. The specificity and abundance of BMP in the endocytic organelles of this early diverged protist suggests that this phospholipid serves a universal and essential function in endocytosis.

Fluorimetric studies of protein kinase C interactions with phospholipids.

Dansyl-phosphatidylserine (D-PS) was used as a fluorescence probe to study interactions between protein kinase C (PKC) with phospholipid vesicles. It was found that D-PS fluorescence (520 nm) was enhanced by PKC (excited at 285 nm). The fluorescence energy transfer, indicative of a close association of PKC with D-PS vesicles, was differentially modulated by various phospholipids, depending upon their effects on PKC activation state and the manners in which they were present. PKC inhibitors (e.g. polymyxin B and ether lipids) potently inhibited the PKC-enhanced D-PS fluorescence. It is suggested that certain spatial arrangements between PKC and its phospholipid cofactor are essential for the enzyme activation and that D-PS would be a useful probe to study fluorimetrically such interactions.

Effect of hyperthermia on lymphocyte plasma membrane. 1. Permeability for glyceraldehyde-3-phosphate dehydrogenase substrates.

The effect of hyperthermia on the permeability of porcine lymphocyte plasma membranes for glyceraldehyde-3-phosphate dehydrogenase (G3PDH) substrates was investigated. The permeability increased in the temperature range of 40-45 degrees C. The temperature dependence for the permeability of G3PDH substrates and for cell viability was not well correlated.