The mannose 6-phosphate receptor and the biogenesis of lysosomes

Localization of the 215 kd mannose 6-phosphate receptor (MPR) was studied in normal rat kidney cells. Low levels of receptor were detected in the trans Golgi network, Golgi stack, plasma membrane, and peripheral endosomes. The bulk of the receptor was localized to an acidic, reticular-vesicular structure adjacent to the Golgi complex. The structure also labeled with antibodies to lysosomal enzymes and a lysosomal membrane glycoprotein (lgp120). While lysosome-like, this structure is not a typical lysosome that is devoid of MPRs. The endocytic marker alpha 2 macroglobulin-gold entered the structure at 37 degrees C, but not at 20 degrees C. With prolonged chase, most of the marker was transported from the structure into lysosomes. We propose that the MPR/lgp-enriched structure is a specialized endosome (prelysosome) that serves as an intermediate compartment into which endocytic vesicles discharge their contents, and where lysosomal enzymes are released from the MPR and packaged along with newly synthesized lysosomal glycoproteins into lysosomes.     

An alternate targeting pathway for procathepsin L in mouse fibroblasts

In transformed mouse fibroblasts, a significant proportion of the lysosomal cysteine protease cathepsin L remains in cells as an inactive precursor which associates with membranes by a mannose phosphate-independent interaction. When microsomes prepared from these cells were resolved on sucrose gradients, this procathepsin L was localized in dense vesicles distinct from those enriched for growth hormone, which is secreted constitutively when expressed in fibroblasts. Ultrastructural studies using antibodies directed against the propeptide to avoid detection of the mature enzyme in lysosomes revealed that the proenzyme was concentrated in dense cores within small vesicles and multivesicular endosomes which labeled with antibodies specific for CD63. Consistent with the resemblance of these cores to those of regulated secretory granules, secretion of procathepsin L from fibroblasts was modestly stimulated by phorbol, 12-myristate, 13-acetate. When protein synthesis was blocked with cycloheximide and lysosomal proteolysis inhibited with leupeptin, procathepsin L was found to gradually convert to the active single-chain protease. The data suggest that when synthesis levels are high, a portion of the procathepsin L is packaged in dense cores within multivesicular endosomes localized near the plasma membrane. Gradual activation of this proenzyme achieves targeting of the proenzyme to lysosomes by a mannose phosphate receptor-independent pathway.     

Distribution and trafficking of MPR300 is normal in cells with cholesterol accumulated in late endocytic compartments: evidence for early endosome-to-TGN trafficking of MPR300

It has been reported that an accumulation of cholesterol within late endosomes/lysosomes in Niemann-Pick type C (NPC) fibroblasts and U18666A-treated cells causes impairment of retrograde trafficking of the cation-independent mannose 6-phosphate/IGF-II receptor (MPR300) from late endosomes to the trans-Golgi network (TGN). In apparent conflict with these results, here we show that as in normal fibroblasts, MPR300 localizes exclusively to the TGN in NPC fibroblasts as well as in normal fibroblasts treated with U18666A. This localization can explain why several lysosomal properties and functions, such as intracellular lysosomal enzyme activity and localization, the biosynthesis of cathepsin D, and protein degradation, are all normal in NPC fibroblasts. These results, therefore, suggest that the accumulation of cholesterol in late endosomes/lysosomes does not affect the retrieval of MPR300 from endosomes to the TGN. Furthermore, treatment of normal and NPC fibroblasts with chloroquine, which inhibits membrane traffic from early endosomes to the TGN, resulted in a redistribution of MPR300 to EEA1 and internalized transferrin-positive, but LAMP-2-negative, early-recycling endosomes. We propose that in normal and NPC fibroblasts, MPR300 is exclusively targeted from the TGN to early endosomes, from where it rapidly recycles back to the TGN without being delivered to late endosomes. This notion provides important insights into the definition of late endosomes, as well as the biogenesis of lysosomes.     

Biogenesis of lysosomes in marshall cells and in cells of the male reproductive system

The mechanism of plasma membrane trafficking and degradation is still poorly understood. This investigation deals with the biogenesis of lysosomes during endocytic flow in Marshall cells and in various cell types of the male reproductive system. Marshall cells were exposed to ammonium chloride (NH4Cl) and leupeptin after labeling with cationic ferritin. In some experiments, the treated cells were immunogold labeled with anti-prosaposin antibody. NH4Cl and leupeptin are lysosomotropic agents that affect the endosomal-lysosomal progression. Testes, efferent ducts and epididymis from mouse mutants with defects affecting plasma membrane degradation were also used to analyze this process. NH4Cl produced a retention of cationic ferritin in endosomes and hindered the endosomal/lysosomal progression. Leupeptin did not affect this process. NH4Cl decreased the labeling of prosaposin in endosomes and lysosomes, while leupeptin increased the labeling of prosaposin in lysosomes. The number of lysosomes per cytoplasmic area was higher in treated cells than in controls. These findings suggest that leupeptin affected lysosomes whereas NH4Cl affected both endosomes and lysosomes. The endosomal and lysosomal accumulation of prosaposin induced by the treatment with NH4Cl and leupeptin indicated that the site of entry of prosaposinwas both the lysosome and endosome. Electron microscopy (EM) of tissues from mouse mutants with defects affecting plasma membrane degradation substantiated these observations. The EM analysis revealed a selective accumulation of multivesicular bodies (MVBs) and the disappearance of lysosomes, in testicular fibroblasts, nonciliated cells of the efferent ducts and principal cells of the epididymis, suggesting that MVBs are precursors of lysosomes. In conclusion: (1) endosomes and MVBs are a required steps for degradation of membranes; (2) endosomes and MVBs are precursors of lysosomes; and (3) endosomes, MVBs, and lysosomes appear to be transient organelles.     

Niemann-Pick type C mutations cause lipid traffic jam

The Niemann–Pick C protein (NPC1) is required for cholesterol transport from late endosomes and lysosomes to other cellular membranes. Mutations in NPC1 cause lysosomal lipid storage and progressive neurological degeneration. Cloning of the NPC1 gene has given us tools with which to investigate the function of this putative cholesterol transporter. Here, we discuss recent studies indicating that NPC1 is not a cholesterol-specific transport molecule. Instead, NPC1 appears to be required for the vesicular shuttling of both lipids and fluid-phase constituents from multivesicular late endosomes to destinations such as the trans -Golgi network.     

Rod/cone dysplasia in Irish setters. Presence of an altered rhodopsin.

The subcellular distribution of beta-glucuronidase acquired by deficient human fibroblasts during co-culture with peritoneal macrophages was compared with that taken up by receptor-mediated endocytosis. Labelled enzyme taken up via receptors was located initially in a low-density endosomal fraction and was transferred to lysosomes within a few minutes. The beta-glucuronidase acquired during 24 h of co-culture was present almost entirely within lysosomes and had a distribution profile identical with that of endogenous beta-hexosaminidase. Monensin prevented transfer of radiolabelled enzyme from endosomes to lysosomes and had a similar effect on the distribution of enzyme acquired by direct transfer, causing beta-glucuronidase to accumulate within endosomes. When the temperature was lowered from 37 degrees C to 19 degrees C, the rate of transfer of enzyme from endosomes to lysosomes was decreased during both direct transfer and indirect receptor-mediated endocytosis. These results show that a lysosomal enzyme acquired by direct transfer during cell-to-cell contact follows a similar intracellular route and has a similar distribution to that of enzymes taken up via cell-surface receptors.     

The lytic granules of natural killer cells are dual-function organelles combining secretory and pre-lysosomal compartments

Cytolytic lymphocytes contain specialized lytic granules whose secretion during cell-mediated cytolysis results in target cell death. Using serial section EM of RNK-16, a natural killer cell line, we show that there are structurally distinct types of granules. Each type is composed of varying proportions of a dense core domain and a multivesicular cortical domain. The dense core domains contain secretory proteins thought to play a role in cytolysis, including cytolysin and chondroitin sulfate proteoglycan. In contrast, the multivesicular domains contain lysosomal proteins, including acid phosphatase, alpha-glucosidase, cathepsin D, and LGP-120. In addition to their protein content, the lytic granules have other properties in common with lysosomes. The multivesicular regions of the granules have an acidic pH, comparable to that of endosomes and lysosomes. The granules take up exogenous cationized ferritin with lysosome-like kinetics, and this uptake is blocked by weak bases and low temperature. The multivesicular domains of the granules are rich in the 270-kD mannose-6-phosphate receptor, a marker which is absent from mature lysosomes but present in earlier endocytic compartments. Thus, the natural killer granules represent an unusual dual-function organelle, where a regulated secretory compartment, the dense core, is contained within a pre-lysosomal compartment, the multivesicular domain.     

Glycoproteins of the lysosomal membrane

Three glycoprotein antigens (120, 100, and 80 kD) were detected by mono- and/or polyclonal antibodies generated by immunization with highly purified rat liver lysosomal membranes. All of the antigens were judged to be integral membrane proteins based on the binding of Triton X-114. By immunofluorescence on normal rat kidney cells, a mouse monoclonal antibody to the 120-kD antigen co-stained with a polyclonal rabbit antibody that detected the 100- and 80-kD antigens as well as with antibodies to acid phosphatase, indicating that these antigens are preferentially localized in lysosomes. Few 120-kD-positive structures were found to be negative for acid phosphatase, suggesting that the antigen was not concentrated in organelles such as endosomes, which lack acid phosphatase. Immunoperoxidase cytochemistry also showed little reactivity in Golgi cisternae, coated vesicles, or on the plasma membrane. Digestion with endo-beta-N-acetylglucosaminidase H (Endo H) and endo-beta-N-acetylglucosaminidase F (Endo F) demonstrated that each of the antigens contained multiple N-linked oligosaccharide chains, most of which were of the complex (Endo H-resistant) type. The 120-kD protein was very heavily glycosylated, having at least 18 N-linked chains. It was also rich in sialic acid, since neuraminidase digestion increased the pI of the 120-kD protein from less than 4 to greater than 8. Taken together, these results strongly suggest that the glycoprotein components of the lysosomal membrane are synthesized in the rough endoplasmic reticulum and terminally glycosylated in the Golgi before delivery to lysosomes. We have provisionally designated these antigens lysosomal membrane glycoproteins lgp120, lgp100, lgp80.     

Rab9 GTPase regulates late endosome size and requires effector interaction for its stability

Rab9 GTPase resides in a late endosome microdomain together with mannose 6-phosphate receptors (MPRs) and the tail-interacting protein of 47 kDa (TIP47). To explore the importance of Rab9 for microdomain establishment, we depleted the protein from cultured cells. Rab9 depletion decreased late endosome size and reduced the numbers of multilamellar and dense-tubule-containing late endosomes/lysosomes, but not multivesicular endosomes. The remaining late endosomes and lysosomes were more tightly clustered near the nucleus, implicating Rab9 in endosome localization. Cells displayed increased surface MPRs and lysosome-associated membrane protein 1. In addition, cells showed increased MPR synthesis in conjunction with MPR missorting to the lysosome. Surprisingly, Rab9 stability on late endosomes required interaction with TIP47. Rabs are thought of as independent, prenylated entities that reside either on membranes or in cytosol, bound to GDP dissociation inhibitor. These data show that Rab9 stability is strongly influenced by a specific effector interaction. Moreover, Rab9 and the proteins with which it interacts seem critical for the maintenance of specific late endocytic compartments and endosome/lysosome localization.     

The membrane composition of coated pits, microvilli, endosomes, and lysosomes is distinctive in the rat kidney proximal tubule cell

The distribution of a number of membrane proteins on plasmalemmal microdomains (microvilli, coated pits) and in endosomes and lysosomes of the proximal tubule epithelial cell was determined in normal rat kidneys by immunofluorescence and immunoelectron microscopy. Two major brush border proteins, 130 and 94 kD, and gamma-glutamyl transpeptidase were detected on the membranes of the microvilli but were not found on membranes of coated pits. Gp330, the Heymann nephritis antigen, and clathrin were localized in coated pits. The lysosomal membrane glycoprotein, lgp120 (Lewis, V., S. A. Green, M. Marsh, P. Vihko, A. Helenius, and I. Mellman, 1985, J. Cell Biol., 100: 1839-1847) was restricted to lysosomes where it co-localized with beta-glucuronidase. Endosomes, identified by preloading with HRP injected 5-15 min before rats were killed, did not contain detectable amounts of any antigen tested. The distribution of the same proteins was also determined in rats given sodium maleate, which is known to slow or reduce protein absorption by the proximal tubule and to cause vacuolation of the endocytic apparatus. After maleate treatment the distribution of microvillar and lysosomal markers was unchanged, but the coated pit markers were redistributed--gp330 was concentrated in newly formed apical vacuoles, and clathrin was diffusely distributed in the apical cytoplasm or on apical coated vesicles. These findings indicate that the membrane composition of microvilli, coated pits, endosomes, and lysosomes is distinctive in the proximal tubule cell; and that gp330, unlike other known coated pit membrane components, is not transferred to endosomes during endocytosis. After maleate treatment, the coated pits lose their clathrin coats, and the corresponding membrane is internalized.     

Sorting of mannose 6-phosphate receptors and lysosomal membrane proteins in endocytic vesicles

The intracellular distributions of the cation-independent mannose 6- phosphate receptor (MPR) and a 120-kD lysosomal membrane glycoprotein (lgp120) were studied in rat hepatoma cells. Using quantitative immunogold cytochemistry we found 10% of the cell's MPR located at the cell surface. In contrast, lgp120 was not detectable at the plasma membrane. Intracellularly, MPR mainly occurred in the trans-Golgi reticulum (TGR) and endosomes. lgp120, on the other hand, was confined to endosomes and lysosomes. MPR was present in both endosomal tubules and vacuoles, whereas lgp120 was confined to the endosomal vacuoles. In cells incubated for 5-60 min with the endocytic tracer cationized ferritin, four categories of endocytic vacuoles could be discerned, i.e., vacuoles designated MPR+/lgp120-, MPR+/lgp120+, MPR-/lgp120+, and vacuoles nonimmunolabeled for MPR and lgp120. Tracer first reached MPR+/lgp120-, then MPR+/lgp120+, and finally MPR-/lgp120+ vacuoles, which are assumed to represent lysosomes. To study the kinetics of appearance of endocytic tracers in MPR-and/or lgp120-containing pools in greater detail, cells were allowed to endocytose horse-radish peroxidase (HRP) for 5-90 min. The reduction in detectability of MPR and lgp120 antigenicity on Western blots, due to treatment of cell homogenates with 3'3-diaminobenzidine, was followed in time. We found that HRP reached the entire accessible pool of MPR almost immediately after internalization of the tracer, while prolonged periods of time were required for HRP to maximally access lgp120. The combined data suggest that MPR+/lgp120+ vacuoles are endocytic vacuoles, intermediate between MPR+/lgp120-endosomes and MPR-/lgp120+ lysosomes, and represent the site where MPR is sorted from lgp120 destined for lysosomes. We propose that MPR is sorted from lgp120 by selective lateral distribution of the receptor into the tubules of this compartment, resulting in the retention of lgp120 in the vacuoles and the net transport of lgp120 to lysosomes.     

Cation-independent mannose 6-phosphate and 78 kDa receptors for lysosomal enzyme targeting are located in different cell compartments

The distribution of the cation-independent mannose 6-phosphate and 78 kDa receptors was studied in postnuclear subcellular fractions from two rat liver cell lines. ELISA assays revealed that the mannose 6-phosphate receptor is enriched in the light buoyant Percoll fractions that contain Golgi structures and early endosomes. Most of the 78 kDa receptor is localized in a heavy fraction at the bottom of the Percoll gradient and smaller amounts in the endosomal fractions. The high-density compartment is denser than lysosomes, contains LAMP2 but not LIMPII or acid hydrolases, and is not disrupted with glycyl-l-phenylalanine 2-naphthylamide, a substrate for cathepsin C that selectively disrupts lysosomes. Immunofluorescence microscopy studies indicate no colocalization of the 78 kDa receptor with the mannose 6-phosphate receptor or LIMPII. Mannose 6-phosphate-independent endocytosed beta-glucuronidase was found in the lysosomal, the early and late endosomal fractions. These fractions were immunoadsorbed in columns containing antibodies against the 78 kDa receptor. Only the endocytosed beta-glucuronidase present in the early and late endosomal fractions is associated to immunoadsorbed vesicles. In these vesicles, LAMP2 was detected but no LIMPII or the mannose 6-phosphate receptor. Results obtained suggest that the 78 kDa receptor is found along the endocytic pathway, but in vesicles different from the cation-independent mannose 6-phosphate receptor.     

An Intralysosomal hsp70 Is Required for a Selective Pathway of Lysosomal Protein Degradation

Previous studies have implicated the heat shock cognate (hsc) protein of 73 kD (hsc73) in stimulating a lysosomal pathway of proteolysis that is selective for particular cytosolic proteins. This pathway is activated by serum deprivation in confluent cultured human fibroblasts. We now show, using indirect immunofluorescence and laser scanning confocal microscopy, that a heat shock protein (hsp) of the 70-kD family (hsp70) is associated with lysosomes (ly-hsc73). An mAb designated 13D3 specifically recognizes hsc73, and this antibody colocalizes with an antibody to lgp120, a lysosomal marker protein. Most, but not all, lysosomes contain ly-hsc73, and the morphological appearance of these organelles dramatically changes in response to serum withdrawal; the punctate lysosomes fuse to form tubules.

Based on susceptibility to digestion by trypsin and by immunoblot analysis after two-dimensional electrophoresis of isolated lysosomes and isolated lysosomal membranes, most ly-hsc73 is within the lysosomal lumen. We determined the functional importance of the ly-hsc73 by radiolabeling cellular proteins with [³H]leucine and then allowing cells to endocytose excess mAb 13D3 before measuring protein degradation in the presence and absence of serum. The increased protein degradation in response to serum deprivation was completely inhibited by endocytosed mAb 13D3, while protein degradation in cells maintained in the presence of serum was unaffected. The intralysosomal digestion of endocytosed [³H]RNase A was not affected by the endocytosed mAb 13D3. These results suggest that ly-hsc73 is required for a step in the degradative pathway before protein digestion within lysosomes, most likely for the import of substrate proteins.

     

Transferrin receptors and cation-independent mannose-6-phosphate receptors deliver their ligands to two distinct subpopulations of multivesicular endosomes

The distribution of transferrin receptors (Tf-R) was determined in Clone 9 hepatocytes and compared to that of 215 kDa, cation-independent mannose-6-phosphate receptors (M6P-R) by double labeling. Cells were allowed to take up exogenous human transferrin (Tf) for 5 to 30 min, after which Tf, Tf-R, and M6P-R were localized by immunofluorescence using specific antibodies. All these proteins were found to be concentrated in the juxtanuclear or Golgi region. When Clone 9 cells were treated with NH4Cl to trap M6P-R in endosomes (Brown, W. J., J. Goodhouse, M. G. Farquhar: J. Cell Biol. 103, 1235-1247 (1986)), the distribution of the two receptors differed: Tf-R remained the same as in controls, but M6P-R were localized in large vacuolated endosomes. To carry out double labeling experiments at the electron microscope level, transferrin gold conjugates (Tf-Au) were prepared, and M6P-R were detected by immunoperoxidase labeling. Tf-Au binding to the cell surface was specific as it was reduced approximately 70 to 79% in the presence of excess native Tf. When Clone 9 cells were incubated with Tf-Au at 37 degrees C for 5 to 30 min, or binding of Tf-Au was carried out at 4 degrees C followed by warming to 37 degrees C, Tf-Au was found within a peripheral tubulovesicular network and within multivesicular endosomes that were not labeled with anti-M6P-R. Other multivesicular endosomes of similar size and morphology were heavily labeled for M6P-R but contained little or no Tf-Au. Tf-Au and M6P-R were also found in separate endosomes in cells treated with NH4Cl. Native Tf was localized in the same compartments as Tf-Au by immunoperoxidase labeling of both Clone 9 cells and mouse myeloma cells. We conclude that in Clone 9 hepatocytes, Tf/Tf-R internalized from the cell surface and M6P-R bearing newly synthesized lysosomal enzymes from the Golgi deliver their ligands to two different subpopulations of multivesicular endosomes. The endosomal subpopulation visited by Tf/Tf-R is known to correspond kinetically to early endosomes. The endosomal subpopulation heavily labeled for M6P-R presumably represent a later endosomal compartment which serves as the junction point where endocytosed ligands and newly synthesized lysosomal enzymes enroute to lysosomes meet.     

Hrs and SNX3 functions in sorting and membrane invagination within multivesicular bodies

After internalization, ubiquitinated signaling receptors are delivered to early endosomes. There, they are sorted and incorporated into the intralumenal invaginations of nascent multivesicular bodies, which function as transport intermediates to late endosomes. Receptor sorting is achieved by Hrs—an adaptor-like protein that binds membrane PtdIns3P via a FYVE motif—and then by ESCRT complexes, which presumably also mediate the invagination process. Eventually, intralumenal vesicles are delivered to lysosomes, leading to the notion that EGF receptor sorting into multivesicular bodies mediates lysosomal targeting. Here, we report that Hrs is essential for lysosomal targeting but dispensable for multivesicular body biogenesis and transport to late endosomes. By contrast, we find that the PtdIns3P-binding protein SNX3 is required for multivesicular body formation, but not for EGF receptor degradation. PtdIns3P thus controls the complementary functions of Hrs and SNX3 in sorting and multivesicular body biogenesis.     

Distribution and structure of the vacuolar H+ ATPase in endosomes and lysosomes from LLC-PK1 cells

Vacuolar proton pumps acidify several intracellular membrane compartments in the endocytic pathway. We have examined the distribution of the vacuolar H+ ATPase in LLC-PK1 cells and the structure of the biosynthetically labeled enzyme in membrane fractions enriched for endosomes or lysosomes. LLC-PK1 cells were allowed to internalize cytochrome c-coated colloidal gold as a marker for endocytic compartments. Proton pumps were identified in these cells by staining the cells with a monoclonal antibody against the vacuolar pump detected with either immunogold or immunoperoxidase techniques. H+ ATPase labeling was seen on structures resembling endosomes and lysosomes, but not on Golgi or plasma membrane. To examine the structure of the H+ ATPase in these compartments, we labeled LLC-PK1 cells for 24 h with [35S]methionine and used a Percoll gradient to obtain fractions enriched for endosomes or lysosomes. H+ ATPase immunoprecipitated from both fractions with monoclonal anti-H+ ATPase antibodies had labeled polypeptides of 70, 56, and 31 kDa. On two-dimensional gels, a comparison of the H+ ATPase from the endosomal and lysosomal fractions revealed that the 70-, 56-, and 31-kDa subunits were similar in both fractions. The results show that the vacuolar H+ ATPase in these cells is distributed primarily in endosomes and lysosomes and that the structure of the enzyme is similar in both compartments.     

Reduction of disulfide bonds within lysosomes is a key step in antigen processing.

Reduction of disulfide bonds is a key step in antigen processing both to allow the unfolding of protein antigens, increasing the access of proteolytic processing enzymes, and to expose free Cys residues within linear peptide epitopes recognized by T cells. We show here that reduction and alkylation of Ag (hen egg lysozyme and ribonuclease A) vastly increased their proteolysis (by specific enzymes or lysosomal fractions) and the production of specific immunogenic peptides that bound to class II MHC molecules recognized by T hybridoma cells. We also show that the lysosome is the vesicular compartment that mediates protein disulfide reduction. We coupled [125I]tyrosine to 131I-alpha 2-macroglobulin or [131I] transferrin via a reducible disulfide linker. Removal of [125I]tyrosine from the alpha 2-macroglobulin conjugate was initiated only after 15 to 20 min of uptake by macrophages, suggesting that reduction occurred late in the endocytic pathway. No reduction of transferrin conjugates was seen, indicating that early, recycling endosomes did not contain reducing activity. Subcellular fractionation showed that the disulfide bonds were reduced only in heavy density (lysosome) fractions and remained intact in fractions of light density (endosomes and plasma membrane). These results indicate the importance of lysosomes in the biochemical processing of protein Ag presented to T cells.     

The Rab7 effector protein RILP controls lysosomal transport by inducing the recruitment of dynein-dynactin motors.

Many intracellular compartments, including MHC class II-containing lysosomes, melanosomes, and phagosomes, move along microtubules in a bidirectional manner and in a stop-and-go fashion due to the alternating activities of a plus-end directed kinesin motor and a minus-end directed dynein-dynactin motor. It is largely unclear how motor proteins are targeted specifically to different compartments. Rab GTPases recruit and/or activate several proteins involved in membrane fusion and vesicular transport. They associate with specific compartments after activation, which makes Rab GTPases ideal candidates for controlling motor protein binding to specific membranes. We and others [7] have identified a protein, called RILP (for Rab7-interacting lysosomal protein), that interacts with active Rab7 on late endosomes and lysosomes. Here we show that RILP prevents further cycling of Rab7. RILP expression induces the recruitment of functional dynein-dynactin motor complexes to Rab7-containing late endosomes and lysosomes. Consequently, these compartments are transported by these motors toward the minus end of microtubules, effectively inhibiting their transport toward the cell periphery. This signaling cascade may be responsible for timed and selective dynein motor recruitment onto late endosomes and lysosomes.     

Re-examination of the Post-translational Arginylated Protein of 125-kD Initially Identified as N-STOP

Post-translational modification of proteins is a complex mechanism by which cells regulate protein activities. One post-translational modification is the incorporation of arginine into the NH2-terminus of proteins. It has been hypothesized that in rat brain extracts, one of the proteins modified by this reaction is the microtubule-associated protein Neuronal Stable Tubule Only Polypeptide (N-STOP). This was inferred from its electrophoretic mobility (125 kD) and because it was immunoprecipitated with a monoclonal antibody against the N-STOP. However, this hypothesis is not supported by our recent results. Herein, we found that rat N-STOP interacts with Ca(2+)-calmodulin, whereas the 125-kD [14C]-arginylated protein does not. The 125-kD [14C]-arginylated protein from rat brain is separated from the N-STOP by two-dimensional electrophoresis, and it is not recognized by a STOP monoclonal antibody. Mouse brain contains N-STOP, which migrates as a protein of 116 kD and could not be labeled by the post-translational incorporation of [14C]-arginine. The 125-kD [14C]-arginylated protein appears in wild-type as well as in STOP knock out mice. Based on these results, we conclude that the 125-kD arginylated protein is different from N-STOP.     

Small GTP-binding proteins on rat liver lysosomal membranes.

GTP-binding proteins have been identified on the membranes of highly purified dextran-filled lysosomes (dextranosomes) and Triton-filled lysosomes (tritosomes) obtained from rat liver. Autoradiography of blots of lysosomal membrane proteins incubated with [alpha-32P]GTP revealed the presence of several specific GTP-binding proteins with a relative molecular mass (M(r)) predominantly in the range of 26-30 kDa. These GTP-binding proteins migrated slower in polyacrylamide gels than purified c-Ha-ras protein expressed in E. coli, whose apparent M(r) was 23 kDa in the same blot. The relative contents of GTP-binding proteins in lysosomal membranes were comparable or greater than that of plasma membranes and of microsomes. Chemical extraction showed that lysosomal GTP-binding proteins were more tightly associated with the membranes than with microsomal GTP-binding proteins. The possible involvement of lysosomal GTP-binding proteins in cellular functions including vacuolar (lysosomal) acidification and organellar dynamics are discussed.