Sphingomyelin is synthesized in the cis Golgi

We have employed in vitro a truncated ceramide analogue with 8 carbon atoms in the sphingosine and the fatty acyl residue, each, to investigate the activity of various membrane fractions to synthesize truncated sphingomyelin. This shortened ceramide readily diffuses through membranes and therefore can easily find access to the lumina of intact organelles. Sphingomyelin synthase activity resides in the Golgi apparatus, and after sucrose density gradient centrifugation of Golgi-enriched fractions sphingomyelin synthesis follows a cis Golgi marker enzyme.     

Golgi Enrichment and Proteomic Analysis of Developing Pinus radiata Xylem by Free-Flow Electrophoresis

Our understanding of the contribution of Golgi proteins to cell wall and wood formation in any woody plant species is limited. Currently, little Golgi proteomics data exists for wood-forming tissues. In this study, we attempted to address this issue by generating and analyzing Golgi-enriched membrane preparations from developing xylem of compression wood from the conifer Pinus radiata. Developing xylem samples from 3-year-old pine trees were harvested for this purpose at a time of active growth and subjected to a combination of density centrifugation followed by free flow electrophoresis, a surface charge separation technique used in the enrichment of Golgi membranes. This combination of techniques was successful in achieving an approximately 200-fold increase in the activity of the Golgi marker galactan synthase and represents a significant improvement for proteomic analyses of the Golgi from conifers. A total of thirty known Golgi proteins were identified by mass spectrometry including glycosyltransferases from gene families involved in glucomannan and glucuronoxylan biosynthesis. The free flow electrophoresis fractions of enriched Golgi were highly abundant in structural proteins (actin and tubulin) indicating a role for the cytoskeleton during compression wood formation. The mass spectrometry proteomics data associated with this study have been deposited to the ProteomeXchange with identifier PXD000557.     

Involvement of Golgi-associated Lyn tyrosine kinase in the translocation of annexin II to the endoplasmic reticulum under oxidative stress

Src-family tyrosine kinases, known to participate in signaling pathways of a variety of receptors at the plasma membrane, are found in cellular endomembranes such as the Golgi apparatus and endosomes. Recently, we showed that Lyn, a member of the Src kinases, accumulates on the Golgi apparatus and then traffics to the plasma membrane. We show here that a majority of endogenous Lyn but not c-Src is accumulated in Golgi-enriched heavy-membrane fractions on a sucrose-density gradient, whereas a small amount of endogenous Lyn is present in light-membrane fractions containing the plasma membrane. Inducible expression of kinase-active Lyn, which biosynthetically reaches the Golgi apparatus, triggers tyrosine phosphorylation of proteins including annexin II. Coimmunoprecipitation analyses reveal that Lyn physically associates with annexin II, and an in vitro kinase assay shows that Lyn phosphorylates annexin II directly. Furthermore, stimulation of cells with H2O2 induces tyrosine phosphorylation of annexin II on the Golgi apparatus in a manner that is dependent on the kinase activity of Src kinases, leading to the translocation of annexin II from the Golgi apparatus to the endoplasmic reticulum. Thus, these results suggest that endomembranes containing the Golgi apparatus where Lyn is anchored can serve as a signaling platform under oxidative stress.     

Src regulates Golgi structure and KDEL receptor-dependent retrograde transport to the endoplasmic reticulum

The tyrosine kinase Src is present on the Golgi membranes. Its role, however, in the overall function and organization of the Golgi apparatus is unclear. We have found that in a cell line called SYF, which lacks the three ubiquitous Src-like kinases (Src, Yes, and Fyn), the organization of the Golgi apparatus is perturbed. The Golgi apparatus is composed of collapsed stacks and bloated cisternae in these cells. Expression of an activated form of Src relocated the KDEL receptor (KDEL-R) from the Golgi apparatus to the endoplasmic reticulum. Other Golgi-specific marker proteins were not affected under these conditions. Because of the specific effect of Src on the location of KDEL-R, we tested whether protein transport between ER and the Golgi apparatus involves Src. Transport of Pseudomonas exotoxin, which is transported to the ER by binding to the KDEL-R is accelerated by inhibition or genetic ablation of Src. Protein transport from ER to the Golgi apparatus however, is unaffected by Src deletion or inhibition. We propose that Src has an appreciable role in the organization of the Golgi apparatus, which may be linked to its involvement in protein transport from the Golgi apparatus to the endoplasmic reticulum.     

Characterization of rat liver endosomal fractions. In vivo activation of insulin-stimulable receptor kinase in these structures

A protocol employing discontinuous sucrose gradient centrifugation was developed to prepare light mitochondrial (L) and Golgi fraction endosomes from simultaneously prepared parent L and microsomal fractions. As judged by the concentration of labeled hormone postinjection, L intermediate and heavy endosome subfractions were 40- to 175-fold purified and Golgi intermediate and heavy endosome subfractions were 30- to 45-fold purified. On electron microscopy, L endosomal fractions contained a predominance of lipoprotein-filled vesicles and were less heterogeneous than corresponding Golgi endosomal fractions. All endosomal fractions were enriched in receptors for insulin and prolactin but binding sites for the former were more broadly distributed in other subfractions than those for the latter. On Percoll gradient centrifugation, L endosomal fractions yielded one peak (rho 1.057) corresponding to the heavier of two peaks seen in Golgi endosomal fractions. The protein composition of high density L and Golgi endosomes, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was similar. The bulk of marker enzymes assayed did not migrate with the endosomal components. Combined acid phosphatase cytochemistry and electron microscope radioautography established that about 80% of the L endosomes contained no acid phosphatase. By affinity labeling and immunological titration with insulin receptor antibody, insulin receptors were identical in L and Golgi endosomes. Insulin-stimulable receptor kinase was demonstrable in both L and Golgi endosome fractions. Following in vivo insulin administration, the insulin receptor kinase in both L and Golgi endosomes was significantly activated. This activated state was not inhibited by a large excess of antiserum to insulin and thus not due to insulin contaminating the partially purified receptor preparation. These observations are compatible with the maintenance and/or initiation of hormone-dependent phosphorylations intracellularly.     

Ephrin-A1 induces c-Cbl phosphorylation and EphA receptor down-regulation in T cells

Eph receptor tyrosine kinases are expressed by T lineage cells, and stimulation with their ligands, the ephrins, has recently been shown to modulate T cell behavior. We show that ephrin-A1 stimulation of Jurkat T cells induces tyrosine phosphorylation of EphA3 receptors and cytoplasmic proteins, including the c-cbl proto-oncogene. Cbl phosphorylation was also observed in peripheral blood T cells. In contrast, stimulation of Jurkat cells with the EphB receptor ligand ephrin-B1 does not cause Cbl phosphorylation. EphA activation also induced Cbl association with Crk-L and Crk-II adapters, but not the related Grb2 protein. Induction of Cbl phosphorylation upon EphA activation appeared to be dependent upon Src family kinase activity, as Cbl phosphorylation was selectively abrogated by the Src family inhibitor 4-amino-5(4-chlorophenyl-7-(tert-butyl)pyrazolo[3,4-d]pyrimidine, while EphA phosphorylation was unimpaired. Ephrin-A1 stimulation of Jurkat cells was also found to cause down-regulation of endogenous EphA3 receptors from the cell surface and their degradation. In accordance with the role of Cbl as a negative regulator of receptor tyrosine kinases, overexpression of wild-type Cbl, but not its 70-Z mutant, was found to down-regulate EphA receptor expression. Receptor down-regulation could also be inhibited by blockage of Src family kinase activity. Our findings show that EphA receptors can actively signal in T cells, and that Cbl performs multiple roles in this signaling pathway, functioning to transduce signals from the receptors as well as regulating activated EphA receptor expression.     

Charge-based separation of detergent-resistant membranes of mouse splenic B cells

Current biochemical characterization for cholesterol- and glycolipid-rich membrane microdomains largely depends on analysis of detergent-resistant membranes (DRMs). In the present study, we succeeded in separation of DRMs of similar density-based on their electrical charge using free-flow electrophoresis (FFE). After crosslinking of B cell receptor (BCR), mouse splenic B cells were lysed with 1% Brij-58 and the resulting lysate was subjected to sucrose density gradient ultracentrifugation. The low-density fraction that recovered a part of DRMs containing IgM together with those enriched in GM1a, the Src family protein tyrosine kinase Lyn, and the alpha subunit of inhibitory heterotrimeric GTP-binding protein was further resolved by FFE. FFE separated the former into more cathodally deflected fractions than the latter. In addition, FFE revealed an anodal shift of DRMs containing a transmembrane protein CD38 upon BCR-crosslinking. The results demonstrate the effectiveness of FFE for the charge-based separation of DRMs.     

Isolation of plasma membrane from amphibian epidermis: evidence for a basal-to-apical charge and activity gradient

Aqueous two-phase partition and preparative free-flow electrophoresis were used in series to isolate the plasma membranes of amphibian epidermis. Fractions obtained by two-phase partition were 40-fold enriched in a K+-stimulated, ouabain-inhibited, p-nitrophenylphosphatase relative to the total homogenate and based on morphology were representative isolates of all epidermal cells together. Small mucosal granules and mucin aggregates were the primary contaminants. Based on activities of marker enzymes, contents of mitochondria, Golgi apparatus and endoplasmic reticulum were low (0.15 that of total homogenate) or absent. When plasma membranes isolated by aqueous two-phase partition were subjected to preparative free-flow electrophoresis, they were distributed toward the anode in a series of fractions of increasing net negative charge, sialic acid content and specific activity of the K+-stimulated, ouabain-inhibited, p-nitrophenylphosphatase reminiscent of the activity gradient from base to apex for frog epidermis observed from cytochemical investigations. The most electronegative fractions nearest the anode and to the left of the main protein peak were enriched in both sulfate groups and thick membranes of the stratum corneum. A fraction migrating less toward the anode and to the right of the main protein peak contained hemidesmosomes together with the lowest enrichments of sialic acid, sulfate and the phosphatase. The results suggest that the plasma membranes isolated from mixed cell populations, such as those encountered in epidermal homogenates, may be resolved by free-flow electrophoresis according to cell type of origin following activity gradients present in the original tissue. Additionally, the findings provide independent biochemical confirmation of a base-to-apex gradient of transport (ATPase) activity associated with the plasma membranes of cells of the different strata of the amphibian epidermis.     

Isolation of Golgi fractions from colchicine-treated rat liver. II. Electrophoretic characterization.

1. Intact Golgi fractions, three from colchicine- or ethanol-treated rat livers and two from a control, were analyzed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. All the fractions showed very similar electrophoretic profiles with 33 protein bands, some of which, especially albumin, had rather higher density in the secretory vesicle fraction than those in the cisternal fraction. 2. Using albumin as the content marker, the Golgi fractions were subfractionated into membranes and contents by freezing-thawing and sonication followed by centrifugation. Distribution of galactosyltransferase among these membrane preparations showed that this enzyme was more enriched in the Golgi cisternal membranes than in the secretory vesicle membranes. 3. All the membrane preparations from the Golgi complex showed very similar patterns on electrophoresis, which were distinctly different from those of microsomal membranes and of plasma membrane. Furthermore, all the Golgi content subfractions had similar protein components, most of which were also found in serum. The microsomal contents, however, showed a considerably different pattern from those of the Golgi contents. 4. From these results it could be concluded that the secretory vesicles are indeed a member of the Golgi complex despite their different appearance and morphology.     

Direct binding of Cbl to Tyr568 and Tyr936 of the stem cell factor receptor/c-Kit is required for ligand-induced ubiquitination, internalization and degradation

The ubiquitin E3 ligase Cbl has been shown to negatively regulate tyrosine kinase receptors, including the stem cell factor receptor/c-Kit. Impaired recruitment of Cbl to c-Kit results in a deregulated positive signalling that eventually can contribute to carcinogenesis. Here, we present results showing that Cbl is activated by the SFKs (Src family kinases) and recruited to c-Kit in order to trigger receptor ubiquitination. We demonstrate that phosphorylated Tyr568 and Tyr936 in c-Kit are involved in direct binding and activation of Cbl and that binding of the TKB domain (tyrosine kinase binding domain) of Cbl to c-Kit is specified by the presence of an isoleucine or leucine residue in position +3 to the phosphorylated tyrosine residue on c-Kit. Apart from the direct association between Cbl and c-Kit, we show that phosphorylation of Cbl by SFK members is required for activation of Cbl to occur. Moreover, we demonstrate that Cbl mediates monoubiquitination of c-Kit and that the receptor is subsequently targeted for lysosomal degradation. Taken together, our findings reveal novel insights into the mechanisms by which Cbl negatively regulates c-Kit-mediated signalling.     

Biosynthesis of the insulin receptor in rat adipose cells. Intracellular processing of the Mr-190 000 pro-receptor.

We investigated the biosynthesis of the insulin receptor in primary cultures of isolated rat adipose cells. Cells were pulse-chase-labelled with [3H]mannose, and at intervals samples were homogenized. Three subcellular membrane fractions were prepared by differential centrifugation: high-density microsomal (endoplasmic-reticulum-enriched), low-density microsomal (Golgi-enriched), and plasma membranes. After detergent solubilization, the insulin receptors were immunoprecipitated with anti-receptor antibodies and analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and autoradiography. After a 30 min pulse-label [3H]mannose first appeared in a band of Mr 190 000. More than 80% of the Mr-190 000 component was recovered in the microsomal fractions. Its intensity reached a maximum at 1 h in the high-density microsomal fraction and at 2 h in the low-density microsomal fraction, and thereafter declined rapidly (t 1/2 approx. 3 h) in both fractions. In the plasma-membrane fraction, the radioactivity in the major receptor subunits, of Mr 135 000 (alpha) and 95 000 (beta), rose steadily during the chase and reached a maximum at 6 h. The Mr-190 000 precursor could also be detected in the high-density microsomal fraction by affinity cross-linking to 125I-insulin. In the presence of monensin, a cationic ionophore that interferes with intracellular transport within the Golgi complex, the processing of the Mr-190 000 precursor into the alpha and beta subunits was completely inhibited. Our results suggest that the Mr-190 000 pro-receptor originates in the endoplasmic reticulum and is subsequently transferred to the Golgi complex. Maturation of the pro-receptor does not seem to be necessary for the expression of the insulin-binding site. Processing of the precursor into the mature receptor subunits appears to occur during the transfer of the pro-receptor from the Golgi complex to the plasma membrane.     

Involvement of cis and trans Golgi apparatus elements in the intracellular sorting and targeting of acid hydrolases to lysosomes

To delineate the traffic route through the Golgi apparatus followed by newly synthesized lysosomal enzymes, we subfractionated the Golgi apparatus of rat liver by preparative free-flow electrophoresis into cisternae fractions of increasing content of trans face markers and decreasing contents of markers for the cis face. NADPase was used to mark median cisternae. Beta-Hexosaminidase, the high mannose oligosaccharide processing enzyme, alpha-mannosidase II, the two enzymes involved in the biosynthesis of the phosphomannosyl recognition marker, and the phosphomannosyl receptor itself decreased in specific activity or amount from cis to trans. Additionally, these activities were observed in a fraction consisting predominantly of cisternae, vesicles and tubules derived from trans-most Golgi apparatus elements. These results, along with preliminary pulse-labeling kinetic data for the phosphomannosyl receptor, suggest that lysosomal enzymes enter the Golgi apparatus at the cis face, are phosphorylated, and appear in trans face vesicles by a route whereby the phosphomannosyl receptor bypasses at least some median and/or trans Golgi apparatus cisternae.     

Activated SRC oncogene phosphorylates R-ras and suppresses integrin activity.

One of the prominent effects of the Src kinase is to reduce cell adhesion. The small GTPase, R-Ras, affects cell adhesion by maintaining integrin activity, and the ability of R-Ras to do so can be regulated by phosphorylation of a tyrosine residue located in its effector domain by an Eph receptor kinase (Zou, J. X., Wang, B., Kalo, M. S., Zisch, A. H., Pasquale, E. B., and Ruoslahti, E. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 13813-13818). Here we show that Src regulates cell adhesion through R-Ras and integrins. Reduced substrate attachment of 293T cells transfected with the cDNA for an activated form of Src (v-Src) was accompanied by phosphorylation of endogenous R-Ras. v-Src also phosphorylated R-Ras in vitro. An activated form of Src similar to one that has been found in human cancers, Src527, shared with v-Src the ability to phosphorylate R-Ras. Stronger R-Ras phosphorylation was seen in Madin-Darby canine kidney cells cells transformed with temperature-sensitive v-Src at the permissive temperature than at the non-permissive temperature, and R-Ras and Src co-immunoprecipitated at the permissive temperature. Mutation analysis showed that the Src phosphorylation site in R-Ras was tyrosine 66, the position critical to the ability of R-Ras to support integrin activity. Finally, activated R-Ras in which tyrosine 66 is mutated to phenylalanine rendered cells partially resistant to the effects of Src on cell adhesion. Regulation of cell adhesion by Src through R-Ras may be at least partially responsible for the reduced adhesion and the resulting increased invasiveness of Src-transformed cells.     

Hck enhances the adherence of lipopolysaccharide-stimulated macrophages via Cbl and phosphatidylinositol 3-kinase

Src family tyrosine kinases have previously been proposed to mediate some of the biological effects of lipopolysaccharide on macrophages. Accordingly, we have sought to identify substrates of Src family kinases in lipopolysaccharide-stimulated macrophages. Stimulation of Bac1.2F5 macrophage cells with lipopolysaccharide was found to induce gradual and persistent tyrosine phosphorylation of Cbl in an Src family kinase-dependent manner. Immunoprecipitation experiments revealed that Cbl associates with Hck in Bac1.2F5 cells, while expression of an activated form of Hck in Bac1.2F5 cells induces tyrosine phosphorylation of Cbl in the absence of lipopolysaccharide stimulation. The Src homology 3 domain of Hck can directly bind Cbl, and this interaction is important for phosphorylation of Cbl. Association of the p85 subunit of phosphatidylinositol (PI) 3-kinase with Cbl is enhanced following lipopolysaccharide stimulation of Bac1.2F5 cells, and transient expression experiments indicate that phosphorylation of Cbl by Hck can facilitate the association of p85 with Cbl. Lipopolysaccharide treatment also stimulates the partial translocation of Hck to the cytoskeleton of Bac1.2F5 cells. Notably, lipopolysaccharide enhances the adherence of Bac1.2F5 cells, an effect that is dependent on the activity of Src family kinases and PI 3-kinase. Thus, we postulate that Hck enhances the adherence of lipopolysaccharide-stimulated macrophages, at least in part, via Cbl and PI 3-kinase.     

Ligand binding induces Cbl-dependent EphB1 receptor degradation through the lysosomal pathway

Eph receptor tyrosine kinases play a critical role in embryonic patterning and angiogenesis. In the adult, they are involved in carcinogenesis and pathological neovascularization. However, the mechanisms underlying their role in tumor formation and metastasis remain to be defined. Here, we demonstrated that stimulation of EphB1 with ephrinB1/Fc led to a marked downregulation of EphB1 protein, a process blocked by the lysosomal inhibitor bafilomycin. Following ephrinB1 stimulation, the ubiquitin ligase Cbl was recruited by EphB1 and then phosphorylated. Both Cbl phosphorylation and EphB1 ubiquitination were blocked by the Src inhibitor PP2. Overexpression of wild-type Cbl, but not of 70Z mutant lacking ligase activity, enhanced EphB1 ubiquitination and degradation. This negative regulation required the tyrosine kinase activity of EphB1 as kinase-dead EphB1-K652R was resistant to Cbl. Glutathione S-transferase binding experiments showed that Cbl bound to EphB1 through its tyrosine kinase-binding domain. In aggregate, we demonstrated that Cbl induces the ubiquitination and lysosomal degradation of activated EphB1, a process requiring EphB1 and Src kinase activity. To our knowledge, this is the first study dissecting the molecular mechanisms leading to EphB1 downregulation, thus paving the way to new means of modulating their angiogenic and tumorigenic properties.     

Subfractionation of rat liver Golgi apparatus by free-flow electrophoresis

Using the technique of preparative free-flow electrophoresis, cisternae of unstacked rat liver Golgi apparatus were separated into a series of fractions of increasing content of sialic acid, thiamine pyrophosphatase and 5'-nucleotidase, markers regarded as being concentrated toward the mature Golgi apparatus face. These same fractions showed a decreasing content of nucleoside diphosphatase, an endoplasmic reticulum marker. Fractions enriched in sialic acid also were enriched in cisternae from the mature or trans face of the Golgi apparatus as deduced from cytochemical criteria. Those fractions least enriched in sialic acid contained cisternae that accumulated deposits of reduced osmium under standard conditions, a test used to mark the opposite, forming or cis-face. Thus subfractionation along the functional polarity axis of the Golgi apparatus with separation of cis and trans face cisternae has been achieved.     

Identification and characterization of Golgi equivalents fromAllomyces macrogynus

Golgi equivalents similar to those described in other fungi were identified in freeze substituted hyphae ofAllomyces macrogynus. These Golgi equivalents were composed of individual or a few loosely associated cisternae, were surrounded by vesicles, and were in a zone relatively free of ribosomes. Certain smooth cisternae in both vegetative hyphae and gametangia stained positively for the Golgi marker enzyme thiamine pyrophosphatase. Subcellular fractionation and biochemical analysis of vegetative hyphae and gametangia revealed endoplasmic reticulum and Golgi membrane fractions with average buoyant densities of 1.09 and 1.15 g/cm³, respectively. Enriched membranes obtained by differential centrifugation were further purified by ultracentrifugation on sucrose step gradients to obtain a presumptive Goldi fraction. Gel electrophoresis of both crude homogenates and fractions prepared by differential centrifugation demonstrated stage-specific glycoproteins that bind the lectin concanavalin A. The results demonstrated thatA. macrogynus has a Golgi complex composed of structurally simple Golgi equivalents and that these Golgi equivalents have physiological functions, such as glycoprotein processing, typically associated with stacked Golgi cisternae from other organisms.     

Membrane flow in plants: Fractionation of growing pollen tubes of tobacco by preparative free-flow electrophoresis and kinetics of labeling of endoplasmic reticulum and golgi apparatus with [3H]leucine

Summary Tobacco (Nicotiana tabacum L.) pollen, germinated 4 hours in suspension culture, was labeled with radioactive leucine and fractionated into constituent membranes by the technique of preparative free-flow electrophoresis. Tubes were ruptured by sonication directly into the electrophoresis buffer. Unfortunately, the Golgi apparatus of the rapidly elongating pollen tubes did not survive the sonication step. However, it was possible to obtain useful fractions of endoplasmic reticulum and mitochondria. To obtain Golgi apparatus, glutaraldehyde was added to the homogenization buffer during sonication. Plasma membrane, which accounted for only about 3% of the total membrane of the homogenates as determined by staining with phosphotungstate at low pH, was obtained in insufficient quantity and fraction purity to permit analysis. Results show rapid incorporation of [³H]leucine into endoplasmic reticulum followed by rapid chase out. The half-time for loss of radioactivity from the pollen tube endoplasmic reticulum was about 10 minutes. Concomitant with the loss of radioactivity from endoplasmic reticulum, the Golgi apparatus fraction was labeled reaching a maximum 20 minutes post chase. The findings suggest flow of membranes from endoplasmic reticulum to the Golgi apparatus during pollen tube growth.     

NADP phosphatase as a marker in free-flow electrophoretic separations for cisternae of the Golgi apparatus midregion

Based on cytochemical analysis, the enzyme NADP phosphatase is most concentrated in the so-called intercalary cisternae from the mid-region of the Golgi apparatus stack. Using free-flow electrophoresis to separate different Golgi regions of rat liver Golgi apparatus, the NADP phosphatase activity, based on estimation of the rate of release of inorganic phosphate from NADP under standard conditions, was similarly localized to membrane fractions from the center of electrophoretic separations. Peak specific activities for both a putative cis marker (NADH-cytochrome c reductase) and an established trans marker (galactosyltransferase) coincided with minima in NADP phosphatase activity, in agreement with the cytochemical observations. The pattern of distribution of enzyme activity for NADP phosphatase differed from that of both acid phosphatase and glucose-6-phosphatase. The pH optimum was 5.0, the K_m for NADP was 0.6 mM and a corresponding production of NAD and inorganic phosphorus was shown. Taken together with other markers for free-flow electrophoresis separation, the NADP phosphatase will provide considerable utility as a specific market to help identify intercalary cisternae of the mammalian Golgi apparatus and to monitor electrophoretic separations.     

Triton-stimulated nucleoside diphosphatase activity: Subcellular localization in corn root homogenates

Summary The distribution of latent UDPase activity (cold storage-activated) is similar to Triton-stimulated UDPase activity in membrane fractions separated by differential centrifugation as well as fractions purified by linear sucrose density centrifugation. The Triton-stimulated UDPase activity appears to be a specific marker for Golgi membranes in corn root homogenates. Detergent-activated UDPase activity provides a more reliable, less cumbersome way to monitor Golgi membranes compared to cold storage-activation and this marker can be used on fresh preparations.This research was supported in part by NSF grant CDP-7927121 and funds received from the Bronfman Science Center, Williams College.