A unique region of RILP distinguishes it from its related proteins in its regulation of lysosomal morphology and interaction with Rab7 and Rab34

Rab7 and Rab34 are implicated in regulation of lysosomal morphology and they share a common effector referred to as the RILP (Rab-interacting lysosomal protein). Two novel proteins related to RILP were identified and are tentatively referred to as RLP1 and RLP2 (for RILP-like protein 1 and 2, respectively). Overexpression of RILP caused enlarged lysosomes that are positioned more centrally in the cell. However, the morphology and distribution of lysosomes were not affected by overexpression of either RLP1 or RLP2. The molecular basis for the effect of RILP on lysosomes was investigated, leading to the demonstration that a 62-residue region (amino acids 272-333) of RILP is necessary for RILP's role in regulating lysosomal morphology. Remarkably, transferring this 62-residue region unique to RILP into corresponding sites in RLP1 rendered the chimeric protein capable of regulating lysosome morphology. A correlation between the interaction with GTP-bound form of both Rab proteins and the capability of regulating lysosomes was established. These results define a unique region in RILP responsible for its specific role in regulating lysosomal morphology as well as in its interaction with Rab7 and Rab34.     

Mutant Huntingtin Impairs Post-Golgi Trafficking to Lysosomes by Delocalizing Optineurin/Rab8 Complex from the Golgi Apparatus

Huntingtin regulates post-Golgi trafficking of secreted proteins. Here, we studied the mechanism by which mutant huntingtin impairs this process. Colocalization studies and Western blot analysis of isolated Golgi membranes showed a reduction of huntingtin in the Golgi apparatus of cells expressing mutant huntingtin. These findings correlated with a decrease in the levels of optineurin and Rab8 in the Golgi apparatus that can be reverted by overexpression of full-length wild-type huntingtin. In addition, immunoprecipitation studies showed reduced interaction between mutant huntingtin and optineurin/Rab8. Cells expressing mutant huntingtin produced both an accumulation of clathrin adaptor complex 1 at the Golgi and an increase of clathrin-coated vesicles in the vicinity of Golgi cisternae as revealed by electron microscopy. Furthermore, inverse fluorescence recovery after photobleaching analysis for lysosomal-associated membrane protein-1 and mannose-6-phosphate receptor showed that the optineurin/Rab8-dependent post-Golgi trafficking to lysosomes was impaired in cells expressing mutant huntingtin or reducing huntingtin levels by small interfering RNA. Accordingly, these cells showed a lower content of cathepsin D in lysosomes, which led to an overall reduction of lysosomal activity. Together, our results indicate that mutant huntingtin perturbs post-Golgi trafficking to lysosomal compartments by delocalizing the optineurin/Rab8 complex, which, in turn, affects the lysosomal function.     

Folliculin directs the formation of a Rab34–RILP complex to control the nutrient‐dependent dynamic distribution of lysosomes

The spatial distribution of lysosomes is important for their function and is, in part, controlled by cellular nutrient status. Here, we show that the lysosome associated Birt–Hoge–Dubé (BHD) syndrome renal tumour suppressor folliculin (FLCN) regulates this process. FLCN promotes the peri‐nuclear clustering of lysosomes following serum and amino acid withdrawal and is supported by the predominantly Golgi‐associated small GTPase Rab34. Rab34‐positive peri‐nuclear membranes contact lysosomes and cause a reduction in lysosome motility and knockdown of FLCN inhibits Rab34‐induced peri‐nuclear lysosome clustering. FLCN interacts directly via its C‐terminal DENN domain with the Rab34 effector RILP. Using purified recombinant proteins, we show that the FLCN‐DENN domain does not act as a GEF for Rab34, but rather, loads active Rab34 onto RILP. We propose a model whereby starvation‐induced FLCN association with lysosomes drives the formation of contact sites between lysosomes and Rab34‐positive peri‐nuclear membranes that restrict lysosome motility and thus promote their retention in this region of the cell.     

Diacylglycerol-activated Hmunc13 serves as an effector of the GTPase Rab34

Hmunc13 is a cytosolic diacylglycerol (DAG)-binding protein, which is upregulated in renal cortical tubule and mesangial cells by hyperglycemia. In response to DAG activation, hmunc13 translocates to the Golgi. To investigate how this may relate to its function, we used a bacterial two-hybrid screen to look for hmunc13-interacting proteins. Full-length Rab34 was specifically isolated from a human kidney cDNA library. Co-expression of the two proteins confirmed Rab34 as a Golgi-associated protein, which was immunoprecipitated from cell lysates by hmunc13. Glutathione S-transferase fusion proteins of WT, Q111L (GTP bound), and T66N (GDP bound) mutants were created, and their GTP-binding activity verified by radioactive overlay assay. Binding of hmunc13 was observed with Q111L, barely detectable with T66N and enhanced with Rab34WT loaded with GTPgammaS compared with GDP loaded. Deletion of munc homolgy domain (MHD)-2, eliminated the hmunc13/Rab34 interaction. The Q111L mutant localized to the Golgi apparatus, but T66N was cytosolic. Localization of both mutants and Rab34WT was unchanged by DAG activation. The data suggest that DAG activation of hmunc13 causes it to be translocated to the Golgi, where it binds to GTP-bound Rab34 via MHD-2. Because Rab34 is known to regulate intracellular lysosome positioning, we propose that hmunc13 serves as an effector of Rab34, mediating lysosome-Golgi trafficking.     

Golgi-bound Rab34 is a novel member of the secretory pathway

Golgi-localized Rab34 has been implicated in repositioning lysosomes and activation of macropinocytosis. Using HeLa cells, we undertook a detailed investigation of Rab34 involvement in intracellular vesicle transport. Immunoelectron microscopy and immunocytochemistry confirmed that Rab34 is localized to the Golgi stack and that active Rab34 shifts lysosomes to the cell center. Contrary to a previous report, we found that Rab34 is not concentrated at membrane ruffles and is not involved in fluid-phase uptake. Also, Rab34-induced repositioning of lysosomes does not affect mannose 6-phosphate receptor trafficking. Most strikingly, HeLa cells depleted of Rab34 by transfection with dominant-negative Rab34 or after RNA interference, failed to transport the temperature-sensitive vesicular stomatitis virus G-protein (VSVG) fused to green fluorescent protein (VSVG-GFP) from the Golgi to the plasma membrane. Transfection with mouse Rab34 rescued this defect. Using endogenous major histocompatibility complex class I (MHCI) as a marker, an endoglycosidase H resistance assay showed that endoplasmic reticulum (ER) to medial Golgi traffic remains intact in knockdown cells, indicating that Rab34 specifically functions downstream of the ER. Further, brefeldin A treatment revealed that Rab34 effects intra-Golgi transport, not exit from the trans-Golgi network. Collectively, these results define Rab34 as a novel member of the secretory pathway acting at the Golgi.     

Overexpression of Rab22a hampers the transport between endosomes and the Golgi apparatus

The transport and sorting of soluble and membrane-associated macromolecules arriving at endosomal compartments require a complex set of Rab proteins. Rab22a has been localized to the endocytic compartment; however, very little is known about the function of Rab22a and inconsistent results have been reported in studies performed in different cell lines. To characterize the function of Rab22a in endocytic transport, the wild-type protein (Rab22a WT), a hydrolysis-deficient mutant (Rab22a Q64L), and a mutant with reduced affinity for GTP (Rab22a S19N) were expressed in CHO cells. None of the three Rab22a constructs affected the transport of rhodamine-dextran to lysosomes, the digestion of internalized proteins, or the lysosomal localization of cathepsin D. In contrast with the mild effect of Rab22a on the endosome-lysosome route, cells expressing Rab22a WT and Rab22a Q64L presented a strong delay in the retrograde transport of cholera toxin from endosomes to the Golgi apparatus. Moreover, these cells accumulated the cation independent mannose 6-phosphate receptor in endosomes. These observations indicate that Rab22a can affect the trafficking from endosomes to the Golgi apparatus probably by promoting fusion among endosomes and impairing the proper segregation of membrane domains required for targeting to the trans-Golgi network (TGN).     

Role of Protein Kinase D in Golgi Exit and Lysosomal Targeting of the Transmembrane Protein,Mcoln1

The targeting of lysosomal transmembrane ( TM ) proteins from the Golgi apparatus to lysosomes is a complex process that is only beginning to be understood. Here, the lysosomal targeting of mucolipin‐1 ( M coln1), the TM protein defective in the autosomal recessive disease, mucolipidosis type IV , was studied by overexpressing full‐length and truncated forms of the protein in human cells, followed by detection using immunofluorescence and immunoblotting. We demonstrated that a 53‐amino acid C ‐terminal region of M coln1 is required for efficient exit from the Golgi . Truncations lacking this region exhibited reduced delivery to lysosomes and decreased proteolytic cleavage of M coln1 into characteristic ～35‐k D a fragments, suggesting that this cleavage occurs in lysosomes. In addition, we found that the co‐expression of full‐length M coln1 with kinase‐inactive protein kinase D ( PKD ) 1 or 2 inhibited M coln1 Golgi exit and transport to lysosomes and decreased M coln1 cleavage. These studies suggest that PKD s play a role in the delivery of some lysosomal resident TM proteins from the Golgi to the lysosomes .     

Functional involvement of TMF/ARA160 in Rab6-dependent retrograde membrane traffic

The small GTPase Rab6 regulates retrograde membrane traffic from endosomes to the Golgi apparatus and from the Golgi to the endoplasmic reticulum (ER). We examined the role of a Rab6-binding protein, TMF/ARA160 (TATA element modulatory factor/androgen receptor-coactivator of 160 kDa), in this process. High-resolution immunofluorescence imaging revealed that TMF signal surrounded Rab6-positive Golgi structures and immunoelectron microscopy revealed that TMF is concentrated at the budding structures localized at the tips of cisternae. The knockdown of either TMF or Rab6 by RNA interference blocked retrograde transport of endocytosed Shiga toxin from early/recycling endosomes to the trans-Golgi network, causing missorting of the toxin to late endosomes/lysosomes. However, the TMF knockdown caused Rab6-dependent displacement of N-acetylgalactosaminyltransferase-2 (GalNAc-T2), but not beta1,4-galactosyltransferase (GalT), from the Golgi. Analyses using chimeric proteins, in which the cytoplasmic regions of GalNAc-T2 and GalT were exchanged, revealed that the cytoplasmic region of GalNAc-T2 plays a crucial role in its TMF-dependent Golgi retention. These observations suggest critical roles for TMF in two Rab6-dependent retrograde transport processes: one from endosomes to the Golgi and the other from the Golgi to the ER.     

A Highlights from MBoC Selection: Coupling of vesicle tethering and Rab binding is required for in vivo functionality of the golgin GMAP-210

Golgins are extended coiled-coil proteins believed to participate in membrane-tethering events at the Golgi apparatus. However, the importance of golgin-mediated tethering remains poorly defined, and alternative functions for golgins have been proposed. Moreover, although golgins bind to Rab GTPases, the functional significance of Rab binding has yet to be determined. In this study, we show that depletion of the golgin GMAP-210 causes a loss of Golgi cisternae and accumulation of numerous vesicles. GMAP-210 function in vivo is dependent upon its ability to tether membranes, which is mediated exclusively by the amino-terminal ALPS motif. Binding to Rab2 is also important for GMAP-210 function, although it is dispensable for tethering per se. GMAP-210 length is also functionally important in vivo. Together our results indicate a key role for GMAP-210–mediated membrane tethering in maintaining Golgi structure and support a role for Rab2 binding in linking tethering with downstream docking and fusion events at the Golgi apparatus.     

Identification of lysosomal and Golgi localization signals in GAP and ARF domains of ARF domain protein 1

ADP ribosylation factors (ARFs) are approximately 20-kDa guanine nucleotide-binding proteins that activate cholera toxin and phospholipase D and are critical components of vesicular trafficking pathways. ARF domain protein 1 (ARD1), a member of the ARF superfamily, contains a 46-kDa amino-terminal extension, which acts as a GTPase-activating protein (GAP) with activity towards its ARF domain. When overexpressed, ARD1 was associated with lysosomes and the Golgi apparatus. In agreement with this finding, lysosomal and Golgi membranes isolated from human liver by immunoaffinity contained native ARD1. ARD1, expressed as a green fluorescent fusion protein, was initially associated with the Golgi network and subsequently appeared on lysosomes, suggesting that ARD1 might undergo vectorial transport between the two organelles. Here we show by microscopic colocalization that GAP and ARF domains determine lysosomal and Golgi localization, respectively, consistent with the presence of more than one signal motif. Using truncated ARD1 molecules, expressed as green fluorescent fusion proteins, it was found that the signal for lysosomal localization was present in residues 301 to 402 of the GAP domain. Site-specific mutagenesis demonstrated that the sequence (369)KXXXQ(373) in the GAP domain was responsible for lysosomal localization. Association of ARD1 with the Golgi apparatus required tyrosine-based motifs. A green fluorescent fusion protein containing the QKQQQQF motif was partially associated with lysosomes, suggesting that this motif contains the information sufficient for lysosomal targeting. These results suggest that ARD1 is a multidomain protein with ARF and GAP regions, which contain Golgi and lysosomal localization signals, respectively, that could function in vesicular trafficking.     

Kinetics of intracellular transport and sorting of lysosomal membrane and plasma membrane proteins

We have used monospecific antisera to two lysosomal membrane glycoproteins, lgp120 and a similar protein, lgp110, to compare the biosynthesis and intracellular transport of lysosomal membrane components, plasma membrane proteins, and lysosomal enzymes. In J774 cells and NRK cells, newly synthesized lysosomal membrane and plasma membrane proteins (the IgG1/IgG2b Fc receptor or influenza virus hemagglutinin) were transported through the Golgi apparatus (defined by acquisition of resistance to endo-beta-N-acetylglucosaminidase H) with the same kinetics (t1/2 = 11-14 min). In addition, immunoelectron microscopy of normal rat kidney cells showed that lgp120 and vesicular stomatitis virus G-protein were present in the same Golgi cisternae demonstrating that lysosomal and plasma membrane proteins were not sorted either before or during transport through the Golgi apparatus. To define the site at which sorting occurred, we compared the kinetics of transport of lysosomal and plasma membrane proteins and a lysosomal enzyme to their respective destinations. Newly synthesized proteins were detected in dense lysosomes (lgp's and beta-glucuronidase) or on the cell surface (Fc receptor or hemagglutinin) after the same lag period (20-25 min), and accumulated at their final destinations with similar kinetics (t1/2 = 30-45 min), suggesting that these two lgp's are not transported to the plasma membrane before reaching lysosomes. This was further supported by measurements of the transport of membrane-bound endocytic markers from the cell surface to lysosomes, which exhibited additional lag periods of 5-15 min and half-times of 1.5-2 h. The time required for transport of newly synthesized plasma membrane proteins to the cell surface, and for the transport of plasma membrane markers from the cell surface to lysosomes would appear too long to account for the rapid transport of lgp's from the Golgi apparatus to lysosomes. Thus, the observed kinetics suggest that lysosomal membrane proteins are sorted from plasma membrane proteins at a post-Golgi intracellular site, possibly the trans Golgi network, before their delivery to lysosomes.     

Rab5a and rab11a mediate agonist-induced trafficking of protease-activated receptor 2

We evaluated the contribution ofrab5a and rab11a to trafficking and signaling of protease-activatedreceptor 2 (PAR2), a receptor for trypsin and tryptase. Agonistsstimulated internalization of PAR2 into early endosomes containingrab5a. Dominant negative rab5aS34N disrupted early endosomes andinhibited agonist-stimulated endocytosis of PAR2. Internalized PAR2 wassorted to lysosomes, and rab5a remained in early endosomes. Rab5apromoted and rab5aS34N impeded resensitization of trypsin-inducedcalcium mobilization. Rab11a was detected in the Golgi apparatus withPAR2, and PAR2 agonists stimulated redistribution of rab11a intovesicles containing PAR2 that migrated to the cell surface. Dominantnegative rab11aS25N was mostly confined to the Golgi apparatus.Although expression of rab11aS25N caused retention of PAR2 in the Golgiapparatus, it did not abolish trafficking of PAR2 to the cell surface.However, expression of wild-type rab11a accelerated both recovery ofPAR2 at the cell surface and resensitization of PAR2 signaling. Thus rab5a is required for PAR2 endocytosis and resensitization, whereas rab11a contributes to trafficking of PAR2 from the Golgi apparatus tothe plasma membrane.

     

The Salmonella effector PipB2 affects late endosome/lysosome distribution to mediate Sif extension

After internalization into mammalian cells, the bacterial pathogen Salmonella enterica resides within a membrane-bound compartment, the Salmonella-containing vacuole (SCV). During its maturation process, the SCV interacts extensively with host cell endocytic compartments, especially late endosomes/lysosomes (LE/Lys) at later stages. These interactions are mediated by the activities of multiple bacterial and host cell proteins. Here, we show that the Salmonella type III effector PipB2 reorganizes LE/Lys compartments in mammalian cells. This activity results in the centrifugal extension of lysosomal glycoprotein-rich membrane tubules, known as Salmonella-induced filaments, away from the SCV along microtubules. Salmonella overexpressing pipB2 induce the peripheral accumulation of LE/Lys compartments, reducing the frequency of LE/Lys tubulation. Furthermore, ectopic expression of pipB2 redistributes LE/Lys, but not other cellular organelles, to the cell periphery. In coexpression studies, PipB2 can overcome the effects of dominant-active Rab7 or Rab34 on LE/Lys positioning. Deletion of a C-terminal pentapeptide motif of PipB2, LFNEF, prevents its peripheral targeting and effect on organelle positioning. The PipB2 homologue PipB does not possess this motif or the same biological activity as PipB2. Therefore, it seems that a divergence in the biological functions of these two effectors can be accounted for by sequence divergence in their C termini.     

The Rab-interacting lysosomal protein, a Rab7 and Rab34 effector, is capable of self-interaction

Rab-interacting lysosomal protein (RILP) has been identified as an interacting partner of the small GTPases Rab7 and Rab34. Active Rab7 recruits RILP on the late endosomal/lysosomal membrane and RILP then functions as a Rab7 effector controlling transport to degradative compartments. Indeed, RILP induces recruitment of dynein-dynactin motor complexes to Rab7-containing late endosomes and lysosomes. Recently, Rab7 and RILP have been found to be key proteins also for the biogenesis of phagolysosomes. Therefore, RILP represents probably an important factor for all endocytic routes to lysosomes. In this study, we show, using the yeast two-hybrid system, that RILP is able to interact with itself. The data obtained with the two-hybrid system were confirmed using co-immunoprecipitation in HeLa cells. The data together indicate that RILP, as already demonstrated for several other Rab effector proteins, is capable of self-association, thus probably forming a homo-dimer.     

LYSOSOMAL PACKAGING IN DIFFERENTIATING AND DEGENERATING ANURAN LATERAL MOTOR COLUMN NEURONS

The role of the Golgi apparatus and the Golgi-endoplasmic reticulum-lysosome complex (GERL) in the genesis of lysosomes was examined in differentiating and degenerating motor neurons of anuran larvae. Acid phosphatase, aryl sulfatase, and thiolacetic acid esterase were utilized as marker enzymes for the lysosomal system, while nucleoside diphosphatase and thiamine pyrophosphatase labeled the inner saccule(s) of the Golgi apparatus. Reduced osmium tetroxide was routinely deposited in the outer Golgi saccule regardless of the state of neuronal maturation. In all young neurons, the disposition of acid hydrolase reaction product paralleled the formation of GERL, with no lytic activity in the Golgi apparatus per se. Hypertrophy of the Golgi apparatus and GERL was observed in the early phases of degeneration, and both organelles apparently exhibit extensive hydrolytic activity. Dense bodies, autophagic vacuoles, and primary lysosomes were found arising from GERL, while the Golgi apparatus may produce primary lysosomal granules during regression. On the other hand, in differentiating neurons, hydrolytic activity was restricted to GERL and an occasional dense body and autophagic vacuole. These studies illustrate a parallelism between the development of GERL and genesis of primary and secondary lysosomes during neuronal cytodifferentiation, and implicate GERL and possibly the Golgi apparatus in lysosomal packaging in degenerating neurons.     

The hypolipidemic compound cetaben induces changes in Golgi morphology and vesicle movement

The human hepatoma cell line HepG2 was used to study the effect of cetaben, a non-fibrate hypolipidemic drug, on cell morphology and vesicle distribution. Cetaben treatment correlated with a fragmentation and/or condensation of Golgi cisternae and the appearance of large electron-lucent vesicles. The Golgi apparatus, demonstrated, for example, by fluorescence-lectin histochemistry, was fragmented after cetaben treatment. The lectin-positive remnants were dispersed throughout the cytoplasm, but with a preference for being transported to tips of cells. However, microtubules and the intermediate filaments as well as the actin microfilaments were unchanged after cetaben treatment indicating that changes in Golgi morphology are not caused by alterations in the cytoskeleton. Cetaben decreases the cholesterol content due to inhibition of cholesterol biosynthesis. Changes in the intracellular cholesterol content are known to influence the intracellular vesicle distribution and are most likely responsible for cetaben-induced Golgi alterations, as depletion of cellular cholesterol by starvation or lovastatin and/or cyclodextrin treatment resulted in a similar redistribution of Golgi-derived wheat germ agglutinin vesicles. These lectin-stained vesicles colocalized with lysosomal marker proteins such as Limp-1 and Lamp-2, but not with the early endosomal markers Rab5 and EEA1. Upon removal of cetaben the lectin- and Limp-1/Lamp-2-costained vesicles dissociated and were transported back to the perinuclear region. Thus, cetaben-induced changes such as fragmentation of the Golgi apparatus and the dispersion of lysosomes away from their juxtanuclear location were reversible.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00418-004-0689-6     

The effector domain of Rab6, plus a highly hydrophobic C terminus, is required for Golgi apparatus localization.

C-terminal lipid modifications are essential for the interaction of Ras-related proteins with membranes. While all Ras proteins are farnesylated and some palmitoylated, the majority of other Ras-related proteins are geranylgeranylated. One such protein, Rab6, is associated with the Golgi apparatus and has a C-terminal CXC motif that is geranylgeranylated on both cysteines. We show here that farnesylation alone cannot substitute for geranylgeranylation in targeting Rab6 to the Golgi apparatus and that whereas Ras proteins that are farnesylated and palmitoylated are targeted to the plasma membrane, mutant Rab proteins that are both farnesylated and palmitoylated associate with the Golgi apparatus. Using chimeric Ras-Rab proteins, we find that there are sequences in the N-terminal 71 amino acids of Rab6 which are required for Golgi complex localization and show that these sequences comprise or include the effector domain. The C-terminal hypervariable domain is not essential for the Golgi complex targeting of Rab6 but is required to prevent prenylated and palmitoylated Rab6 from localizing to the plasma membrane. Functional analysis of these mutant Rab6 proteins in Saccharomyces cerevisiae shows that wild-type Rab6 and C-terminal mutant Rab6 proteins which localize to the Golgi apparatus in mammalian cells can complement the temperature-sensitive phenotype of ypt6 null mutants. Interestingly, therefore, the C-terminal hypervariable domain of Rab6 is not required for this protein to function in S. cerevisiae.     

The mannose 6-phosphate receptor: function,biosynthesis and translocation

This report summarizes studies concerning the role of the lysosomal protein: Man-6-P receptor and describes some recent data on its biosynthesis and cellular translocation. The receptor functions both in the Golgi apparatus (or GERL) and on the cell surface where it binds lysosomal proteins and mediates their transport to lysosomes. Consistent with its dual role, the receptor in several cell types has been localized to the plasma membrane and Golgi cisternae, to clathrin-coated structures at both locations, and to vesicles characteristic of endosomes or CURL. Biosynthetic studies have shown that the receptor undergoes several post-translational modifications including the processing of its asparagine-linked oligosaccharides, phosphorylation of serine residues, and unknown modifications required for acquisition of immunoreactivity and functional activity. Cellular pools of mature receptor readily mix as evidenced by rapid labeling of intracellular receptor by exogenously added receptor antibodies. Degradation of the receptor occurs non-lysosomally and is perhaps mediated by extracellular Man-6-P-containing hydrolases. A working hypothesis for the mechanism of Man-6-P receptor function that is consistent with these observations is presented.     

Ultrastructural distribution of NADPase within the Golgi apparatus and lysosomes of mammalian cells

Cytochemical studies with over 40 different mammalian cell types have indicated that NADPase activity is associated with the Golgi apparatus and/or lysosomes of all cells. In the majority of cases, NADPase is restricted to saccular elements comprising the medial region of the Golgi stack and an occasional lysosome. There is often weak NADPase activity in other Golgi compartments such as the trans Golgi saccules and/or elements of the trans Golgi network. In some cells, however, strong NADPase activity is found within these latter compartments, either exclusively in trans Golgi saccules or elements of the trans Golgi network, or in combination with medial Golgi saccules and each other including (1) medial Golgi saccules + trans Golgi saccules, (2) medial Golgi saccules + trans Golgi saccules + trans Golgi network, or (3) trans Golgi saccules + trans Golgi network. In some rare cases, no NADPase activity is detectable in either Golgi saccules or elements of the trans Golgi network, but it is observed in an occasional lysosome or throughout the lysosomal system of these cells. It is unclear at present if these variations in the distribution of NADPase across the Golgi apparatus, and between the Golgi apparatus and lysosomal system, are due to differences in targeting mechanisms or to the existence of "bottlenecks" in the natural flow of NADPase along the biosynthetic pathway toward lysosomes. While no clear pattern in the association of strong NADPase activity with lysosomes was apparent relative to the ultrastructural distribution of NADPase activity in Golgi saccules or elements of the trans Golgi network, the results of this investigation suggested that cells having NADPase localized predominantly toward the trans aspect of the Golgi apparatus (in trans Golgi saccules or elements of the trans Golgi network or both) have few NADPase-positive lysosomes. The only exception is hepatocytes which were classified as predominantly trans but had noticeable NADPase activity within medial Golgi saccules and elements of the trans Golgi network as well, and highly reactive lysosomes. Other cells showing highly reactive lysosomes including (1) Kupffer cells of liver and those forming the proximal convoluted tubules of the kidney, both of which also had strong NADPase activity within medial and trans Golgi saccules and elements of the trans Golgi network, (2) Leydig cells of the testis and interstitial cells of the ovary, which also showed strong NADPase activity within medial Golgi saccules, and (3) macrophages from lung, spleen and testis, and Sertoli cells from the testis all of which showed no Golgi associated NADPase activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Fine structure of the lysosomes in the two types of synoviocytes of normal rat synovial membrane

Summary The lysosomal system of the two types of synoviocytes (A and S) from the knee joint of normal rat synovial membrane was studied by electron-microscopic acid phosphatase cytochemistry. In random sections of the synovial intima lysosomes were more often encountered in the A-cell profiles than in the S-cell profiles. Characteristically, type-A synoviocytes showed many large and medium-sized lysosomes the cytochemical appearance of which varied considerably. No acid phosphatase activity was detectable in the cisternae of the Golgi apparatus or in the Golgi vesicles. In type-S synoviocytes the lysosomes were smaller, and more uniform in cytochemical appearance. Heavy deposits of acid phosphatase reaction product were constantly demonstrated in cisternae of the Golgi apparatus as well as in smooth-walled Golgi vesicles in type-S cells. The findings that type-A and type-S synoviocytes show distinctly different organization of the lysosomal system indicate that the roles of the lysosomes in these two types of cells may be different.