Nucleocytoplasmic traffic of proteins.

We have used the synchronized formation of a mixed cytoplasm upon heterokaryon formation as a model for investigating the cisternal-specific transport of resident proteins between neighboring Golgi apparatus. Rat NRK and hamster 15B cells were fused by UV-inactivated Sindbis virus and then incubated for various time periods in the presence of cycloheximide. The resident Golgi apparatus proteins, rat GIMPc and Golgp 125, were localized with species-specific monoclonal antibodies. Immunofluorescent colocalization of rat and hamster Golgi membrane proteins was observed with a t1/2 of 1.75 h at 37 degrees C. Colocalization of resident, but not transient, Golgi membrane protein was concomitant with formation of a large extended Golgi complex and was accompanied by the acquisition of endoglycosidase H resistance by preexisting Golgp 125. Dispersal of the extended Golgi complex by nocodazole revealed that colocalization of resident Golgi proteins was due to intermixing of proteins in the same Golgi element rather than overlapping of closely apposed Golgi structures. Incubation of the polykaryons at 20 degrees C inhibited both the colocalization of GIMPc and Golgp 125 and the formation of an extended Golgi complex. Little change in the number of cisternae/stack in cross sections of the Golgi apparatus was observed upon cell fusion, and in the extended Golgi complex the hamster resident protein remained localized to one side of the Golgi stack. Surprisingly, the morphological identity of the rat and hamster Golgi units appeared to be maintained in the heterokaryons. These results suggest that the intermixing of resident Golgi membrane proteins requires direct physical continuity between Golgi elements and that resident Golgi membrane proteins are preferentially excluded from the non-clathrin-coated transport vesicles budding from Golgi cisternae.     

Antibodies to rat pancreas Golgi subfractions: identification of a 58- kD cis-Golgi protein

A 58-kD cis-Golgi protein has been identified by generating polyclonal antibodies against heavy (cis) Golgi subfractions. Total microsomes isolated from rat pancreatic homogenates were subfractionated to yield a rough microsomal fraction (B1) and three smooth membrane subfractions (B2-B4) enriched in cis-, middle, and trans-Golgi elements, respectively. The heavy (cis) subfraction, B2 (d = 1.17 g/ml), was fractionated by Triton X-114 phase separation, and the proteins recovered in the detergent phase were used to immunize rabbits. One of the anti-B2 antibodies obtained gave a "Golgi"-staining pattern when screened by immunofluorescence on normal rat kidney cells and mouse RPC 5.4 myeloma cells. In rat pancreatic exocrine cells the antibody reacted with the plasmalemma as well as elements in the Golgi region. By immunoelectron microscopy, the antigen recognized by anti-B2 IgG was found to be restricted to cis-Golgi elements in myeloma cells where it was concentrated in the fenestrated cis-most cisterna and in some of the tubules and vesicles located along the cis face of the Golgi complex. By immunoprecipitation and immunoblotting, the anti-B2 IgG exclusively recognized a 58-kD protein in myeloma cells. The anti-B2 IgG reacted with several proteins in solubilized pancreatic B2 membranes, including a 58-kD protein, but affinity-purified anti-58-kD IgG reacted exclusively with the 58-kD protein. These results suggest that the 58-kD protein is a specific component of cis-Golgi membranes.     

Monoclonal antibodies as markers of the endocytic and secretory pathways

A galactosyltransferase-rich subcellular fraction and wheat germ agglutinin(WGA)-binding microsomal proteins from rat myeloma cells have been used to immunize BALB/c mice. Fusion of the corresponding spleen cells with the Sp2/0 mouse myeloma has lead to the production of hybridomas secreting monoclonal antibodies directed against four proteins of the Golgi complex (GC) and other smooth membranes (SM). Subcellular fractionation of myeloma cells and rat liver, Triton X-114 partitioning, protease treatment and lectin binding studies have permitted us to identify--by immunoblotting--the molecular weight of the proteins involved, their topology and their mode of association with membranes. Morphological analysis has been performed by immunocytochemistry at the light and electron microscopic level. Judging by these criteria, the GCII antigen is a protein of 44 kDa which is loosely associated with the endodomain of Golgi cisternae. GCIII is a detergent-binding glycoprotein of 130 kDa whose epitope is on the endodomain of Golgi cisternae. SMI is a detergent-binding glycoprotein of 58 to 90 kDa found at several stations along the endocytic path: in coated pits, coated vesicles, endocytic vesicles, but not in lysosomes. The epitope recognized by the corresponding antibody faces the ectodomain. When this antibody is added to living cells in culture, it is rapidly internalized. SMII is a detergent-binding glycoprotein of 140 kDa. The epitope recognized is restricted to membranes of Golgi complex cisternae and multivesicular bodies. These reagents should be useful for dissection and perturbation of vesicular traffic.     

Identification of the mouse beta'-COP Golgi component as a spermatocyte autoantigen in scleroderma and mapping of its gene Copb2 to mouse chromosome 9

In an immunological screening of a mouse testicular cDNA library with a human CREST serum we isolated five overlapping cDNA clones encoding the mouse homolog of a Golgi coatomer complex protein (accession number AF043120), designated beta'-COP in bovine and p102 in humans. We generated antibodies against this protein which specifically recognize the Golgi apparatus of mouse spermatocytes. FISH analyses assigned the beta'-COP gene Copb2 to mouse Chromosome 9, region E3-F1. Our results demonstrate that CREST sera can contain antibody components against Golgi proteins as well as against nuclear proteins.     

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.     

Immunocytochemical localization of amylase and chymotrypsinogen in the exocrine pancreatic cell with special attention to the Golgi complex

Affinity-purified, monospecific rabbit antibodies against rat pancreatic alpha-amylase and bovine pancreatic alpha-chymotrypsinogen were used for immunoferritin observations of ultrathin frozen sections of mildly fixed exocrine pancreatic tissue from secretion-stimulated (pilocarpine) rats and from overnight-fasted rats and guinea pigs. The labeling patterns for both antibodies were qualitatively alike: Labeling occurred in (a) the cisternae of the rough endoplasmic reticulum (RER) including the perinuclear cisterna, in (b) the peripheral area between the RER and cis-Golgi face, and (c) all Golgi cisternae, condensing vacuoles, and secretory granules. Labeling of cytoplasmic matrix was negligible. Structures that appeared to correspond to rigid lamellae were unlabeled. Differences in labeling intensities indicated that concentration of the zymogens starts at the boundary of the RER and cis-side of the Golgi complex. These data support the view that the Golgi cisternae are involved in protein processing in both stimulated and unstimulated cells and that Golgi cisternae and condensing vacuoles constitute a functional unit.     

Immunocytochemical localization of BiP to the rough endoplasmic reticulum: evidence for protein sorting by selective retention

Immunoglobulin heavy chain binding protein (BiP) (also known as GRP 78) is a protein of the endoplasmic reticulum (ER) which has been shown to be involved in post-translational processing of nascent membrane and secretory proteins. To determine BiP's location in the exocytic pathway, we localized BiP at the electron microscopic level in mouse myeloma cell lines by immunoperoxidase cytochemistry. BiP was found to be present within the cisternal spaces of the RER and nuclear envelope but was not detected in the cisternae of the Golgi complex. BiP reaction product was also found within transitional elements of the RER but was absent from smooth-surfaced vesicles found between the ER and the Golgi complex. Immunoperoxidase staining of BiP was reduced or absent in regions of a smooth ER membrane system in myelomas that contained endogenous murine retrovirus A particles. All compartments of the exocytic pathway, including the virus-containing smooth ER, stained for IgG, a secretory protein. These observations suggest that BiP is selectively retained in the cisternae of the ER and is not free to enter Golgi-directed transport vesicles. These studies suggest that BiP's subcellular localization may occur by selective interaction with component(s) of the ER.     

Immunocytochemical visualization of the Golgi apparatus in several species, including human, and tissues with an antiserum against MG-160, a sialoglycoprotein of rat Golgi apparatus

We used a monoclonal antibody (10A8), derived from mice immunized with fractions enriched in Golgi apparatus of rat brain neurons, to isolate an intrinsic membrane sialoglycoprotein of 160 KD from rat brain. By immunoelectron microscopy the sialoglycoprotein, named MG-160, was localized in medical cisternae of the Golgi apparatus of neurons, glia, adenohypophysis, and cultured rat pheochromocytoma (PC 12). The monoclonal antibody (MAb) reacted only with rat tissues. Because the epitope(s) recognized by a monoclonal antibody may be restricted, localization of an antigen by a single MAb may not reflect the extent of the distribution of antigen in various species and tissues. Therefore, to further investigate the presence and localization of MG-160 or of an antigenically related protein in several species and tissues, we used a polyclonal antiserum raised against MG-160 purified by antibody (10A8) affinity chromatography. Immunoblots of crude microsomal fractions from rat brain probed with the antiserum against MG-160 showed two to three prominent bands of approximately 160, 150, and 68 KD. Immunoblots of crude microsomal fractions from human, chicken, and frog brains showed prominent bands of 130-140 and 68 KD. Immunoblots of crude membrane fractions from Saccharomyces cerevisiae showed prominent bands of approximately 110-120 and 80 KD. Light microscopic immunocytochemical studies with frog, chicken, mouse, rat, rabbit, bovine, and human brains and with several other rat and human tissues showed a staining pattern consistent with the Golgi apparatus. Immunoelectron microscopy with rat and human brain and with rat myocardium and pituitary showed prominent and exclusive staining of cis, medial, and occasionally trans cisternae of the Golgi apparatus. The cisternae of the trans Golgi network were not stained. These findings are consistent with the hypothesis that a polypeptide related to MG-160 is present in the Golgi apparatus of several tissues in human, rodents, chicken, and frog and possibly in Saccharomyces cerevisiae. The antiserum to MG-160 represents a reliable reagent for immunohistochemical visualization of the Golgi apparatus in brain and several other human tissues obtained at autopsy, fixed with Bouin's, and embedded in paraffin.     

Characterization of the Golgi Complex Cleared of Proteins in Transit and Examination of Calcium Uptake Activities

To characterize endogenous molecules and activities of the Golgi complex, proteins in transit were >99% cleared from rat hepatocytes by using cycloheximide (CHX) treatment. The loss of proteins in transit resulted in condensation of the Golgi cisternae and stacks. Isolation of a stacked Golgi fraction is equally efficient with or without proteins in transit [control (CTL SGF1) and cycloheximide (CHX SGF1)]. Electron microscopy and morphometric analysis showed that >90% of the elements could be positively identified as Golgi stacks or cisternae. Biochemical analysis showed that the cis-, medial-, trans-, and TGN Golgi markers were enriched over the postnuclear supernatant 200- to 400-fold with and 400- to 700-fold without proteins in transit. To provide information on a mechanism for import of calcium required at the later stages of the secretory pathway, calcium uptake into CTL SGF1 and CHX SGF1 was examined. All calcium uptake into CTL SGF1 was dependent on a thapsigargin-resistant pump not resident to the Golgi complex and a thapsigargin-sensitive pump resident to the Golgi. Experiments using CHX SGF1 showed that the thapsigargin-resistant activity was a plasma membrane calcium ATPase isoform in transit to the plasma membrane and the thapsigargin-sensitive pump was a sarcoplasmic/endoplasmic reticulum calcium ATPase isoform. In vivo both of these calcium ATPases function to maintain millimolar levels of calcium within the Golgi lumen.     

Identification of a 200-kD, brefeldin-sensitive protein on Golgi membranes

A mAb AD7, raised against canine liver Golgi membranes, recognizes a novel, 200-kD protein (p200) which is found in a wide variety of cultured cell lines. Immunofluorescence staining of cultured cells with the AD7 antibody produced intense staining of p200 in the juxtanuclear Golgi complex and more diffuse staining of p200 in the cytoplasm. The p200 protein in the Golgi complex was colocalized with other Golgi proteins, including mannosidase II and beta-COP, a coatomer protein. Localization of p200 by immunoperoxidase staining at the electron microscopic level revealed concentrations of p200 at the dilated rims of Golgi cisternae. Biochemical studies showed that p200 is a peripheral membrane protein which partitions to the aqueous phase of Triton X-114 solutions and is phosphorylated. The p200 protein is located on the cytoplasmic face of membranes, since it was accessible to trypsin digestion in microsomal preparations, and is recovered in approximately equal amounts in membrane pellets and in the cytosol of homogenized cells. Immunofluorescence staining of normal rat kidney cells exposed to the toxin brefeldin A (BFA), showed that there was very rapid redistribution of p200, which was dissociated from Golgi membranes in the presence of this drug. The effect of BFA was reversible, since upon removal of the toxin, AD7 rapidly reassociated with the Golgi complex. In the BFA-resistant cell line PtK1, BFA failed to cause redistribution of p200 from Golgi membranes. Taken together, these results indicate that the p200 Golgi membrane-associated protein has many properties in common with the coatomer protein, beta-COP.     

Reinternalization of secretory proteins during membrane recycling in rat pancreatic acinar cells

Membrane recycling in pancreatic acinar cells involves endocytic vesicle formation at the apical cell surface and rapid membrane traffic to the Golgi complex. During this process a small amount of extracellular content is taken up from the acinar lumen. In order to determine whether secretory proteins already released into the pancreatic acinar lumen are reinternalized during membrane retrieval, 3H-labeled amylase or 125I-labeled secretory proteins were reinfused through the pancreatic duct until the lumina were reached. Tissue samples from various time points were prepared for light and electron microscope autoradiography. The observations showed that [3H]amylase and, to a lesser extent, the 125I-labeled secretory proteins were internalized at the apical cell surface and rapidly (within 2-5 min) transferred to the Golgi cisternae and the condensing vacuoles; only a minor proportion of silver grains was observed over lysosomes. In addition, at later time points, mature secretion granules close to the Golgi complex became labeled. The results indicate that exocytosis in the rat exocrine pancreas does not operate at 100% efficiency; part of the exported amylase and part of the total secretion product are reinternalized concomitantly with the endocytic removal of plasma membrane and are copackaged together with newly synthesized secretory proteins.     

Characterization of cytoplasmically oriented Golgi proteins with a monoclonal antibody

BALB/c mice were repeatedly immunized with a galactosyl transferase- rich microsomal fraction of rat myeloma cells. Spleen cells were subsequently fused with Sp2/0 mouse myeloma cells, the resulting hybridomas were cloned, and their secreted Ig was screened for reactivity with antigens belonging to the Golgi complex. One such monoclonal antibody, 6F4C5, gave especially intense immunofluorescent staining of the Golgi area of myeloma cells and fibroblasts. It recognized two proteins bands on immunoblots of gel-fractionated cell lysates: a major one with an estimated Mr of 54,000 and a minor one at 86,000. Both proteins were concentrated in microsomal fractions isolated at low ionic strength. They were hydrophilic judging from partitioning of a Triton X-114 cell lysate. Both were cytoplasmically oriented as demonstrated by protease and high KCl treatments of postmitochondrial supernatants and microsomal fractions. Neither was retained by columns of insolubilized wheat germ agglutinin or concanavalin A, which suggests that they are not glycoproteins. Their more detailed location in the Golgi complex was studied by immunoelectron microscopy, using a saponin permeabilization procedure and peroxidase-conjugated reagents. The observed staining was restricted to two or three cisternae in the medial part of the stack. Nevertheless, differential centrifugation experiments indicated that the two antigens may be recovered in distinct subcellular fractions: this may be related to the unexpected observation that rather low salt concentrations strip the antigens from microsomal fraction.     

Cisterna-specific localization of glycosylation-related proteins to the Golgi apparatus

The Golgi apparatus is an intracellular organelle playing central roles in post-translational modification and in the secretion of membrane and secretory proteins. These proteins are synthesized in the endoplasmic reticulum (ER) and transported to the cis-, medial-and trans-cisternae of the Golgi. While trafficking through the Golgi, proteins are sequentially modified with glycan moieties by different glycosyltransferases. Therefore, it is important to analyze the glycosylation function of the Golgi at the level of cisternae. Markers widely used for cis-, medial- and trans-cisternae/trans Golgi network (TGN) in Drosophila are GM130, 120 kDa and Syntaxin16 (Syx16); however the anti-120 kDa antibody is no longer available. In the present study, Drosophila Golgi complex-localized glycoprotein-1 (dGLG1) was identified as an antigen recognized by the anti-120 kDa antibody. A monoclonal anti-dGLG1 antibody suitable for immunohistochemistry was raised in rat. Using these markers, the localization of glycosyltransferases and nucleotide-sugar transporters (NSTs) was studied at the cisternal level. Results showed that glycosyltransferases and NSTs involved in the same sugar modification are localized to the same cisternae. Furthermore, valuable functional information was obtained on the localization of novel NSTs with as yet incompletely characterized biochemical properties.     

Transferrin receptors recycle to cis and middle as well as trans Golgi cisternae in Ig-secreting myeloma cells

The recycling itinerary of plasma membrane transferrin receptors (TFR) was charted in IgG-secreting mouse myeloma cells (RPC 5.4) by tagging surface receptors with either bound anti-transferrin receptor antibodies (anti-TFR) or Fab fragments thereof and determining the intracellular destinations of the tagged receptors by immunocytochemistry. By immunofluorescence, TFR tagged with either probe were seen to be rapidly internalized and translocated from the cell surface to the juxtanuclear (Golgi) region. When localized by immunoperoxidase procedures at the electron microscopic level, the anti-TFR-labeled receptors were detected in all cisternae (cis, middle, and trans) of the Golgi stacks as well as in endosomes and trans Golgi reticular elements. There was no difference in the routing of TFR tagged with monovalent Fab and those tagged with divalent IgG. Tagged receptors were detected in Golgi stacks of approximately 50% of the cells analyzed. The position of the labeled cisternae within a given stack was found to be quite variable with cis and middle cisternae more often labeled at 5 min and trans cisternae at 30 min of antibody uptake. The finding that recycling plasmalemmal TFR can visit all or most Golgi subcompartments raises the likely possibility that any Golgi-associated posttranslational modification can occur during recycling as well as during the initial biosynthesis of plasmalemma receptors and other membrane proteins.     

Molecular mechanism of mitotic Golgi disassembly and reassembly revealed by a defined reconstitution assay

In mammalian cells, flat Golgi cisternae closely arrange together to form stacks. During mitosis, the stacked structure undergoes a continuous fragmentation process. The generated mitotic Golgi fragments are distributed into the daughter cells, where they are reassembled into new Golgi stacks. In this study, an in vitro assay has been developed using purified proteins and Golgi membranes to reconstitute the Golgi disassembly and reassembly processes. This technique provides a useful tool to delineate the mechanisms underlying the morphological change. There are two processes during Golgi disassembly: unstacking and vesiculation. Unstacking is mediated by two mitotic kinases, cdc2 and plk, which phosphorylate the Golgi stacking protein GRASP65 and thus disrupt the oligomer of this protein. Vesiculation is mediated by the COPI budding machinery ARF1 and the coatomer complex. When treated with a combination of purified kinases, ARF1 and coatomer, the Golgi membranes were completely fragmented into vesicles. After mitosis, there are also two processes in Golgi reassembly: formation of single cisternae by membrane fusion, and restacking. Cisternal membrane fusion requires two AAA ATPases, p97 and NSF (N-ethylmaleimide-sensitive fusion protein), each of which functions together with specific adaptor proteins. Restacking of the newly formed Golgi cisternae requires dephosphorylation of Golgi stacking proteins by the protein phosphatase PP2A. This systematic study revealed the minimal machinery that controls the mitotic Golgi disassembly and reassembly processes.     

The Golgi complex. III. The effects of puromycin on ultrastructure and glycoprotein synthesis.

The effect of puromycin has been investigated on protein and glycoprotein synthesis and on ultrastructure of the Golgi complex from rat liver. Incorporation of [14C]leucine into protein in Golgi fractions and into serum proteins was depressed rapidly after puromycin treatment. In the serum proteins, incorporation returned to normal levels at 2 h whereas in Golgi fractions it continued to rise to 200% of the control levels at 3 h and was still elevated at 24 h after puromycin treatment. Incorporation of [14C]glucosamine into glycoprotein was depressed in Golgi and serum fractions in a similar manner but slightly later than that of leucine. Leucine labelled material found at 3 h was a poor acceptor for carbohydrate, since [14C]glucosamine incorporation was not elevated above control values. Galactosyl transferase activity was not depressed in the Golgi membranes and, at 3 h, was elevated implying that an adequate supply of enzyme was available at all times. The activity of the galactosyl transferase in serum appeared to be depressed suggesting that transport of enzyme from Golgi complex to serum was defective. Ultrastructural changes in the Golgi complex were observed to occur rapidly after puromycin treatment. The cisternae became irregular, compressed, and degenerated progressively from central region towards the periphery. Irregular tubular structures formed at the expense of cisternal membrane and showed accumulation of low density lipoprotein. Vesiculation and degenerative changes of the Golgi membranes continued from 2-12 h while more typical arrangements of the Golgi complex were observed between 24-48 h. The morphological changes correlated with changes in glycoprotein synthesis.     

Got1p and Sft2p: membrane proteins involved in traffic to the Golgi complex.

Traffic through the yeast Golgi complex depends on a member of the syntaxin family of SNARE proteins, Sed5p, present in early Golgi cisternae. Sft2p is a non-essential tetra-spanning membrane protein, found mostly in the late Golgi, that can suppress some sed5 alleles. We screened for mutations that show synthetic lethality with sft2 and found one that affects a previously uncharacterized membrane protein, Got1p, as well as new alleles of sed5 and vps3. Got1p is an evolutionarily conserved non-essential protein with a membrane topology similar to that of Sft2p. Immunofluorescence and subcellular fractionation indicate that it is present in early Golgi cisternae. got1 mutants, but not sft2 mutants, show a defect in an in vitro assay for ER-Golgi transport at a step after vesicle tethering to Golgi membranes. In vivo, inactivation of both Got1p and Sft2p results in phenotypes ascribable to a defect in endosome-Golgi traffic, while their complete removal results in an ER-Golgi transport defect. Thus the presence of either Got1p or Sft2p is required for vesicle fusion with the Golgi complex in vivo. We suggest that Got1p normally facilitates Sed5p-dependent fusion events, while Sft2p performs a related function in the late Golgi.     

Phospholipase D2 localizes to the plasma membrane and regulates angiotensin II receptor endocytosis

Phospholipase D (PLD) is a key facilitator of multiple types of membrane vesicle trafficking events. Two PLD isoforms, PLD1 and PLD2, exist in mammals. Initial studies based on overexpression studies suggested that in resting cells, human PLD1 localized primarily to the Golgi and perinuclear vesicles in multiple cell types. In contrast, overexpressed mouse PLD2 was observed to localize primarily to the plasma membrane, although internalization on membrane vesicles was observed subsequent to serum stimulation. A recent report has suggested that the assignment of PLD2 to the plasma membrane is in error, because the endogenous isoform in rat secretory cells was imaged and found to be present primarily in the Golgi apparatus. We have reexamined this issue by using a monoclonal antibody specific for mouse PLD2, and find, as reported initially using overexpression studies, that endogenous mouse PLD2 is detected most readily at the plasma membrane in multiple cell types. In addition, we report that mouse, rat, and human PLD2 when overexpressed all similarly localize to the plasma membrane in cell lines from all three species. Finally, studies conducted using overexpression of wild-type active or dominant-negative isoforms of PLD2 and RNA interference-mediated targeting of PLD2 suggest that PLD2 functions at the plasma membrane to facilitate endocytosis of the angiotensin II type 1 receptor.     

GRASP55, a second mammalian GRASP protein involved in the stacking of Golgi cisternae in a cell-free system.

We have identified a 55 kDa protein, named GRASP55 (Golgi reassembly stacking protein of 55 kDa), as a component of the Golgi stacking machinery. GRASP55 is homologous to GRASP65, an N-ethylmaleimide-sensitive membrane protein required for the stacking of Golgi cisternae in a cell-free system. GRASP65 exists in a complex with the vesicle docking protein receptor GM130 to which it binds directly, and the membrane tethering protein p115, which also functions in the stacking of Golgi cisternae. GRASP55 binding to GM130, could not be detected using biochemical methods, although a weak interaction was detected with the yeast two-hybrid system. Cryo-electron microscopy revealed that GRASP65, like GM130, is present on the cis-Golgi, while GRASP55 is on the medial-Golgi. Recombinant GRASP55 and antibodies to the protein block the stacking of Golgi cisternae, which is similar to the observations made for GRASP65. These results demonstrate that GRASP55 and GRASP65 function in the stacking of Golgi cisternae.