A quantitative radioimmunological screening method for specific gene products

A rapid, sensitive, and quantitative radioimmunoassay for specific translation products was developed using Escherichia coli cells grown in 96-well microtiter plates. A simple and inexpensive apparatus that facilitates the simultaneous transfer of all 96 detergent-lysed cultures to nitrocellulose within 30 s is described. Following this quantitative transfer, selected proteins are screened using specific antisera and ¹²⁵I-Protein A. The technique works with either cytoplasmic or membrane proteins. As little as a two- to threefold increase of a gene product over normal levels can easily be detected.     

Sites of sulfatation in the chondrocytes of the articular cartilage of the rabbit

35S sulfate uptake by the articular cartilage chondrocytes, from biopsies of rabbit, have been studied by high resolution autoradiography. The Golgi apparatus, rough endoplasmic reticulum, cytosol, cytoplasmic membrane and extracellular space were considered as cell compartments in the quantitative analysis of the autoradiograms. The results obtained show: 1) a high activity of radiosotope incorporation in the Golgi apparatus; 2) a fast rhythm of transfer of the substances labelled in the Golgi apparatus to the cell membrane; 3) significant labelling of the rough endoplasmic reticulum, throughout the experiment. It is concluded: 1) The grains observed in the rough endoplasmic reticulum show a significant radioisotope uptake on this level, and this evidence some sulfotransferase activity. 2) The high 35S sulfate uptake level which is observed in the Golgi apparatus demonstrates that the highest sulfotransferase enzyme activity is located in this cell area, thus showing that the "early" sulfation that began in the rough endoplasmic reticulum was completed by a "late" sulfation in the Golgi apparatus. It is here that complete chondromucoprotein building takes place before being excreted. 3) The high transfer level of the labelled substances from the Golgi apparatus shows that the sulfated product secretion for building the cartilage matrix takes place rapidly since a great label increase can be already observed at the beginning of the chase period in the outer surrounding area of the chondrocyte membrane.     

Is cytochrome P-450 transported from the endoplasmic reticulum to the Golgi apparatus in rat hepatocytes?

The Golgi apparatus mediates intracellular transport of not only secretory and lysosomal proteins but also membrane proteins. As a typical marker membrane protein for endoplasmic reticulum (ER) of rat hepatocytes, we have selected phenobarbital (PB)-inducible cytochrome P- 450 (P-450[PB]) and investigated whether P-450(PB) is transported to the Golgi apparatus or not by combining biochemical and quantitative ferritin immunoelectron microscopic techniques. We found that P-450(PB) was not detectable on the membrane of Golgi cisternae either when P-450 was maximally induced by phenobarbital treatment or when P-450 content in the microsomes rapidly decreased after cessation of the treatment. The P-450 detected biochemically in the Golgi subcellular fraction can be explained by the contamination of the microsomal vesicles derived from fragmented ER membranes to the Golgi fraction. We conclude that when the transfer vesicles are formed by budding on the transitional elements of ER, P-450 is completely excluded from such regions and is not transported to the Golgi apparatus, and only the membrane proteins destined for the Golgi apparatus, plasma membranes, or lysosomes are selectively collected and transported.     

Decoupling Polarization of the Golgi Apparatus and GM1 in the Plasma Membrane

Cell polarization is a process of coordinated cellular rearrangements that prepare the cell for migration. GM1 is synthesized in the Golgi apparatus and localized in membrane microdomains that appear at the leading edge of polarized cells, but the mechanism by which GM1 accumulates asymmetrically is unknown. The Golgi apparatus itself becomes oriented toward the leading edge during cell polarization, which is thought to contribute to plasma membrane asymmetry. Using quantitative image analysis techniques, we measure the extent of polarization of the Golgi apparatus and GM1 in the plasma membrane simultaneously in individual cells subject to a wound assay. We find that GM1 polarization starts just 10 min after stimulation with growth factors, while Golgi apparatus polarization takes 30 min. Drugs that block Golgi polarization or function have no effect on GM1 polarization, and, conversely, inhibiting GM1 polarization does not affect Golgi apparatus polarization. Evaluation of Golgi apparatus and GM1 polarization in single cells reveals no correlation between the two events. Our results indicate that Golgi apparatus and GM1 polarization are controlled by distinct intracellular cascades involving the Ras/Raf/MEK/ERK and the PI3K/Akt/mTOR pathways, respectively. Analysis of cell migration and invasion suggest that MEK/ERK activation is crucial for two dimensional migration, while PI3K activation drives three dimensional invasion, and no cumulative effect is observed from blocking both simultaneously. The independent biochemical control of GM1 polarity by PI3K and Golgi apparatus polarity by MEK/ERK may act synergistically to regulate and reinforce directional selection in cell migration.     

C11ORF24 Is a Novel Type I Membrane Protein That Cycles between the Golgi Apparatus and the Plasma Membrane in Rab6-Positive Vesicles

The Golgi apparatus is an intracellular compartment necessary for post-translational modification, sorting and transport of proteins. It plays a key role in mitotic entry through the Golgi mitotic checkpoint. In order to identify new proteins involved in the Golgi mitotic checkpoint, we combine the results of a knockdown screen for mitotic phenotypes and a localization screen. Using this approach, we identify a new Golgi protein C11ORF24 (NP_071733.1). We show that C11ORF24 has a signal peptide at the N-terminus and a transmembrane domain in the C-terminal region. C11ORF24 is localized on the Golgi apparatus and on the trans-Golgi network. A large part of the protein is present in the lumen of the Golgi apparatus whereas only a short tail extends into the cytosol. This cytosolic tail is well conserved in evolution. By FRAP experiments we show that the dynamics of C11ORF24 in the Golgi membrane are coherent with the presence of a transmembrane domain in the protein. C11ORF24 is not only present on the Golgi apparatus but also cycles to the plasma membrane via endosomes in a pH sensitive manner. Moreover, via video-microscopy studies we show that C11ORF24 is found on transport intermediates and is colocalized with the small GTPase RAB6, a GTPase involved in anterograde transport from the Golgi to the plasma membrane. Knocking down C11ORF24 does not lead to a mitotic phenotype or an intracellular transport defect in our hands. All together, these data suggest that C11ORF24 is present on the Golgi apparatus, transported to the plasma membrane and cycles back through the endosomes by way of RAB6 positive carriers.     

Both post-Golgi and intra-Golgi cycling affect the distribution of the Golgi phosphoprotein GPP130

Golgi phosphoprotein, GPP130, a cis Golgi protein, is representative of proteins cycling between the Golgi apparatus and endosomes in a pH-sensitive manner. The present qualitative data are insufficient to distinguish the relative contributions of Golgi and endosomal processes in regulating the cycling of such proteins. We have taken a quantitative approach to analyze GPP130 distribution in response to pH perturbation. We have used Shiga-like toxin B fragment, a protein that traffics from the cell surface and Golgi apparatus by the late endosomal bypass pathway, as a probe to highlight one aspect of GPP130 cycling and similarly the trafficking of tsO45-green fluorescent protein (GFP) between the Golgi apparatus and the plasma membrane to treat that aspect of GPP130 cycling in isolation. Overall, we conclude from quantitative analysis and simulations that treatment of HeLa cells with the pH perturbant, monensin, affects GPP130 cycling at several stages with effects on (i) intra-Golgi cycling, (ii) trans Golgi to endosome transport and (iii) endosome to Golgi transport. Our analysis indicates that the effect is greatest at the trans Golgi, the most acidic portion of the Golgi apparatus. In sum, multiple, regulated steps affect the trafficking of GPP130.     

Structural differentiation of membranes involved in the secretion of polysaccharide slime by root cap cells of cress (Lepidium sativum L.)

The peripheral secretion tissue of the root cap of Lepidium sativum L. was investigated by electronmicroscopy and freeze-fracturing in order to study structural changes of membranes involved in the secretion process of polysaccharide slime. Exocytosis of slime-transporting vesicles occurs chiefly in the distal region of the anticlinal cell walls. The protoplasmic fracture face (PF) of the plasmalemma of this region is characterized by a high number of homogenously distributed intramembranous particles (IMPs) interrupted by areas nearly free of IMPs. Near such areas slime-transporting vesicles are found to be underlying the plasma membrane. It can be concluded that areas poor in particles are prospective sites for membrane fusion. During the formation of slime-transporting vesicles, the number of IMPs undergoes a striking change in the PF of dictyosome membranes and their derivatives. It is high in dictyosome cisternae and remarkably lower in the budding region at the periphery of the cisternae. Slime-transporting vesicles are as poor in IMPs as the areas of the plasmalemma. Microvesicles rich in IMPs are observed in the surroundings of dictyosomes. The results indicate that in the plasmalemma and in membranes of the Golgi apparatus special classes of proteins — recognizable as IMPs — are displaced laterally into adjacent membrane regions. Since the exoplasmic fracture face (EF) of these membranes is principally poor in particles, it can be concluded that membrane fusion occurs in areas characterized by a high quantity of lipid molecules. It is obvious that the Golgi apparatus regulates the molecular composition of the plasma membrane by selection of specific membrane components. The drastic membrane transformation during the formation of slime-transporting vesicles in the Golgi apparatus causes the enrichment of dictyosome membranes by IMPs, whereas the plasma membrane probably is enriched by lipids. The structural differentiations in both the plasma membrane and in Golgi membranes are discussed in relation to membrane transformation, membrane flow, membrane fusion, and recycling of membrane constituents.Abbreviations PF protoplasmic fracture face - EF exoplasmic fracture face - IMP intramembranous particle     

Isolation of plasma membrane,Golgi apparatus,and endoplasmic reticulum fractions from single homogenates of mouse liver

Procedures to isolate plasma membrane, Golgi apparatus, and endoplasmic reticulum from a single homogenate of mouse liver are described. Fractions contain low levels of contaminating membranes as determined from morphometry and analyses of marker enzymes. The method requires only 2–3 gm of liver as starting material and yields approximately 0.7, 0.7, and 0.5 mg protein/gm liver, respectively, for endoplasmic reticulum, Golgi apparatus, and plasma membrane. Golgi apparatus fractions show high levels of galactosyltransferase activity and consist of cisternal stacks and associated secretory vesicles and tubules. Endoplasmic reticulum fractions are enriched in both glucose-6-phosphatase and nicotinamide adenine dinucleotide phosphate (reduced) (NADPH)-cytochrome c reductase and contain membrane vesicles with attached ribosomes. K⁺-stimulated p-nitrophenyl phosphatase and (Na⁺ K⁺) adenosine triphosphatase activity are enriched in the plasma membrane fraction. This fraction consists of membrane sheets, many with junctional complexes, and bile canaliculi that are representative of the total hepatocyte plasma membrane. The fractionation procedure is designed to utilize small amounts of tissue (e.g., with liver slices), to reduce the total time required for fractionation, and to permit comparisons of constituents of plasma membrane, Golgi apparatus, and endoplasmic reticulum prepared from the same starting homogenates.     

Subcellular biosynthesis and transport of gangliosides formed from exogenous lactosylceramide in rat liver.

In order to clarify the mechanisms of ganglioside biosynthesis and transport we intravenously administered a liposomal dispersion of radiolabelled lactosylceramide (LacCer) to rats and then followed the time course of the individual gangliosides which became radioactive in the Golgi-apparatus and plasma-membrane fractions prepared from the liver. After administration of radiolabelled LacCer the liver retained a substantial amount of radioactivity, which was distributed among an organic phase (mainly residual LacCer), a fraction containing low-Mr substances (mainly 3H2O) and a ganglioside fraction. The hepatocytes were found to provide the bulk of gangliosides biosynthesized from exogenous LacCer. After subcellular fractionation, the total radioactive gangliosides increased in the Golgi apparatus up to 8 h, to then decrease and practically disappear at 24 h; in the plasma membranes they were progressively concentrated, accounting for high absolute values. Ganglioside patterns were greatly modified with time in both the Golgi apparatus and plasma membrane, but without significant differences between them. Biosynthesis in the Golgi apparatus and accumulation in the plasma membrane of each individual ganglioside followed a precursor-product relationship. The obtained results indicated that once a ganglioside is biosynthesized in the Golgi apparatus, it is in part made available for translocation to the plasma membrane, which rapidly occurs, and is in part retained in the Golgi apparatus, where it acts as a precursor for the biosynthesis of more glycosylated gangliosides.     

Biogenesis of peroxisomes. Topogenesis of the peroxisomal membrane and matrix proteins

Genetic and proteomic approaches have led to the identification of 32 proteins, collectively called peroxins, which are required for the biogenesis of peroxisomes. Some are responsible for the division and inheritance of peroxisomes; however, most peroxins have been implicated in the topogenesis of peroxisomal proteins. Peroxisomal membrane and matrix proteins are synthesized on free ribosomes in the cytosol and are imported post-translationally into pre-existing organelles (Lazarow PB & Fujiki Y (1985) Annu Rev Cell Biol1, 489-530). Progress has been made in the elucidation of how these proteins are targeted to the organelle. In addition, the understanding of the composition of the peroxisomal import apparatus and the order of events taking place during the cascade of peroxisomal protein import has increased significantly. However, our knowledge on the basic principles of peroxisomal membrane protein insertion or translocation of peroxisomal matrix proteins across the peroxisomal membrane is rather limited. The latter is of particular interest as the peroxisomal import machinery accommodates folded, even oligomeric, proteins, which distinguishes this apparatus from the well characterized translocons of other organelles. Furthermore, the origin of the peroxisomal membrane is still enigmatic. Recent observations suggest the existence of two classes of peroxisomal membrane proteins. Newly synthesized class I proteins are directly targeted to and inserted into the peroxisomal membrane, while class II proteins reach their final destination via the endoplasmic reticulum or a subcompartment thereof, which would be in accord with the idea that the peroxisomal membrane might be derived from the endoplasmic reticulum.     

Maintenance of Golgi structure and function depends on the integrity of ER export.

The Golgi apparatus comprises an enormous array of components that generate its unique architecture and function within cells. Here, we use quantitative fluorescence imaging techniques and ultrastructural analysis to address whether the Golgi apparatus is a steady-state or a stable organelle. We found that all classes of Golgi components are dynamically associated with this organelle, contrary to the prediction of the stable organelle model. Enzymes and recycling components are continuously exiting and reentering the Golgi apparatus by membrane trafficking pathways to and from the ER, whereas Golgi matrix proteins and coatomer undergo constant, rapid exchange between membrane and cytoplasm. When ER to Golgi transport is inhibited without disrupting COPII-dependent ER export machinery (by brefeldin A treatment or expression of Arf1[T31N]), the Golgi structure disassembles, leaving no residual Golgi membranes. Rather, all Golgi components redistribute into the ER, the cytoplasm, or to ER exit sites still active for recruitment of selective membrane-bound and peripherally associated cargos. A similar phenomenon is induced by the constitutively active Sar1[H79G] mutant, which has the additional effect of causing COPII-associated membranes to cluster to a juxtanuclear region. In cells expressing Sar1[T39N], a constitutively inactive form of Sar1 that completely disrupts ER exit sites, Golgi glycosylation enzymes, matrix, and itinerant proteins all redistribute to the ER. These results argue against the hypothesis that the Golgi apparatus contains stable components that can serve as a template for its biogenesis. Instead, they suggest that the Golgi complex is a dynamic, steady-state system, whose membranes can be nucleated and are maintained by the activities of the Sar1-COPII and Arf1-coatomer systems.     

Direct Pathway from Early/Recycling Endosomes to the Golgi Apparatus Revealed through the Study of Shiga Toxin B-fragment Transport

Shiga toxin and other toxins of this family can escape the endocytic pathway and reach the Golgi apparatus. To synchronize endosome to Golgi transport, Shiga toxin B-fragment was internalized into HeLa cells at low temperatures. Under these conditions, the protein partitioned away from markers destined for the late endocytic pathway and colocalized extensively with cointernalized transferrin. Upon subsequent incubation at 37°C, ultrastructural studies on cryosections failed to detect B-fragment–specific label in multivesicular or multilamellar late endosomes, suggesting that the protein bypassed the late endocytic pathway on its way to the Golgi apparatus. This hypothesis was further supported by the rapid kinetics of B-fragment transport, as determined by quantitative confocal microscopy on living cells and by B-fragment sulfation analysis, and by the observation that actin- depolymerizing and pH-neutralizing drugs that modulate vesicular transport in the late endocytic pathway had no effect on B-fragment accumulation in the Golgi apparatus. B-fragment sorting at the level of early/recycling endosomes seemed to involve vesicular coats, since brefeldin A treatment led to B-fragment accumulation in transferrin receptor–containing membrane tubules, and since B-fragment colocalized with adaptor protein type 1 clathrin coat components on early/recycling endosomes. Thus, we hypothesize that Shiga toxin B-fragment is transported directly from early/recycling endosomes to the Golgi apparatus. This pathway may also be used by cellular proteins, as deduced from our finding that TGN38 colocalized with the B-fragment on its transport from the plasma membrane to the TGN.     

Four-dimensional imaging of filter-grown polarized epithelial cells

Understanding how epithelial cells generate and maintain polarity and function requires live cell imaging. In order for cells to become fully polarized, it is necessary to grow them on a permeable membrane filter; however, the translucent filter obstructs the microscope light path required for quantitative live cell imaging. Alternatively, the membrane filter may be excised but this eliminates selective access to apical and basolateral surfaces. Conversely, epithelial cells cultured directly on glass exhibit different phenotypes and functions from filter grown cells. Here, we describe a new method for culturing polarized epithelial cells on a Transwell filter insert that allows superior live cell imaging with spatial and temporal image resolution previously unachievable using conventional methods. Cells were cultured on the underside of a filter support. Epithelial cells grown in this inverted configuration exhibit a fully polarized architecture, including the presence of functional tight junctions. This new culturing system permits four-dimensional (three spatial dimension over time) imaging of endosome and Golgi apparatus dynamics, and permits selective manipulation of the apical and basolateral surfaces. This new technique has wide applicability for visualization and manipulation of polarized epithelial cells.     

Signal-anchor sequences are an essential factor for the Golgi-plasma membrane localization of type II membrane proteins

Despite studies of the mechanism underlying the intracellular localization of membrane proteins, the specific mechanisms by which each membrane protein localizes to the endoplasmic reticulum, Golgi apparatus, and plasma membrane in the secretory pathway are unclear. In this study, a discriminant analysis of endoplasmic reticulum, Golgi apparatus and plasma membrane-localized type II membrane proteins was performed using a position-specific scoring matrix derived from the amino acid propensity of the sequences around signal-anchors. The possibility that the sequence around the signal-anchor is a factor for identifying each localization group was evaluated. The discrimination accuracy between the Golgi apparatus and plasma membrane-localized type II membrane proteins was as high as 90%, indicating that, in addition to other factors, the sequence around signal-anchor is an essential component of the selection mechanism for the Golgi and plasma membrane localization. These results may improve the use of membrane proteins for drug delivery and therapeutic applications.     

CERT-mediated trafficking of ceramide

The transport and sorting of lipids from the sites of their synthesis to their appropriate destinations are fundamental for membrane biogenesis. In the synthesis of sphingolipids in mammalian cells, ceramide is newly produced at the endoplasmic reticulum (ER), and transported from the ER to the trans Golgi regions, where it is converted to sphingomyelin. CERT mediates the ER-to-Golgi trafficking of ceramide. CERT contains several functional domains and motifs including i) a START domain capable of catalyzing inter-membrane transfer of ceramide, ii) a pleckstrin homology domain, which serves to target the Golgi apparatus, iii) a FFAT motif which interacts with the ER-resident membrane protein VAP, and iv) a serine-repeat motif, of which hyperphosphorylation down-regulates CERT activity. It has been suggested that CERT extracts ceramide from the ER and carries it to the Golgi apparatus in a non-vesicular manner and that efficient CERT-mediated trafficking of ceramide occurs at membrane contact sites between the ER and the Golgi apparatus.     

Targeting of proteins to the Golgi apparatus

The Golgi apparatus maintains a highly organized structure in spite of the intense membrane traffic which flows into and out of this organelle. Resident Golgi proteins must have localization signals to ensure that they are targeted to the correct Golgi compartment and not swept further along the secretory pathway. There are a number of distinct groups of Golgi membrane proteins, including glycosyltransferases, recyclingtrans-Golgi network proteins, peripheral membrane proteins, receptors and viral glycoproteins. Recent studies indicate that there are a number of different Golgi localization signals and mechanisms for retaining proteins to the Golgi apparatus. This review focuses on the current knowledge in this field.     

The FliP and FliR proteins of Salmonella typhimurium, putative components of the type III flagellar export apparatus, are located in the flagellar basal body

Most of the structural components of the flagellum of Salmonella typhimurium are exported through a flagellum-specific pathway, which is a member of the family of type III secretory pathways. The export apparatus for this process is poorly understood. A previous study has shown that two proteins, about 23 and 26 kDa in size and of unknown genetic origin, are incorporated into the flagellar basal body at a very early stage of flagellar assembly. In the present study, we demonstrate that these basal body proteins are FliP (in its mature form after signal peptide cleavage) and FliR respectively. Both of these proteins have homologues in other type III secretion systems. By placing a FLAG epitope tag on FliR and the MS-ring protein FliF and immunoblotting isolated hook basal body complexes with anti-FLAG monoclonal antibody, we estimate (using the FLAG-tagged FliF as an internal reference) that the stoichiometry of FliR is fewer than three copies per basal body. An independent estimate of stoichiometry was made using data from an earlier quantitative radiolabelling analysis, yielding values of around four or five subunits per basal body for FliP and around one subunit per basal body for FliR. Immunoelectron microscopy using anti-FLAG antibody and gold–protein A suggests that FliR is located near the MS ring. We propose that the flagellar export apparatus contains FliP and FliR and that this apparatus is embedded in a patch of membrane in the central pore of the MS ring.     

Are the photosynthetic membranes of cryptophyte algae inside out?

Summary Cryptomonads are unicellular algae with a unique photosynthetic apparatus, both in structure and pigment composition. A cryptomonad,Rhodomonas lens (R. lens), was studied by conventional electron microscopy, freeze-fracture, and freeze-etch in order to determine whether the thylakoids of this alga are everted with respect to those of other plants, as has been postulated (Gantt et al. 1971,Gantt 1979, 1980) as a means to compensate for the location of the cryptomonad light-harvesting apparatus on the opposite side of the thylakoid membrane from that of related algae. We have characterized the thylakoids of this alga and conclude that they are not everted, but are oriented in the same manner as those of other algae and green plants. Implications for energy transfer are discussed.     

Post-Golgi apparatus localization and regional expression of rat intestinal sialyltransferase detected by immunoelectron microscopy with polypeptide epitope-purified antibody

During studies on the Golgi apparatus immunolocalization of beta-galactoside alpha 2,6-sialyltransferase in intestinal cells, immunostaining of a number of post-Golgi apparatus structures including mucus droplets and plasma membrane were observed. In order to determine if this labeling was in fact due to sialyltransferase and not carbohydrate-specific antibodies in the polyclonal antiserum preparation, fusion protein to sialyltransferase was used to epitope purify polypeptide-specific antibodies. The affinity purification was performed on a column containing a beta-galactosidase-sialyltransferase fusion protein expressed in Escherichia coli. Using such antibodies we present evidence that in intestinal cells sialyltransferase is not only present in the Golgi apparatus cisternal stack but also its transtubular network and various post-Golgi apparatus structures. In absorptive enterocytes, post-Golgi apparatus vesicles, the brush border and basolateral plasma membrane, multivesicular bodies, and lysosome-like structures were labeled. In goblet cells the limiting membrane and lumen of forming and mature mucus droplets as well as the plasma membrane exhibited label for sialyltransferase. The results provide evidence for "ecto-sialyltransferase" in the plasma membranes of these cells, and suggest that most of the sialyltransferase is released from the Golgi membranes and becomes secreted with the goblet cell mucus. In addition, the polypeptide epitope-purified antibody was also used to examine regional expression of sialyltransferase in the rat intestinal epithelium. Immunolabel was restricted to the large intestine and not found in duodenum, jejunum, and ileum. Direct measurement of the enzyme activity was found to correlate with the immunoelectron microscopic data. This observation suggests that there is regional specific expression of the beta-galactoside alpha 2,6-sialyltransferase.     

Oligomerization and intracellular protein transport: dimerization of intestinal dipeptidylpeptidase IV occurs in the Golgi apparatus.

It was postulated that newly synthesized membrane proteins need to be assembled into oligomers in the endoplasmic reticulum in order to be transported to the Golgi apparatus. By use of the differentiated human adenocarcinoma cell line Caco-2, the general validity of this proposal was studied for small intestinal brush border enzymes which are dimers in most mammalian species. Chemical cross-linking experiments and sucrose gradient rate-zonal centrifugation revealed that dipeptidylpeptidase IV is present as a dimer in the brush border membrane of Caco-2 cells whereas the disaccharidase sucrase-isomaltase appears to be a monomer. Dipeptidylpeptidase IV was found to dimerize immediately after complex glycosylation, an event associated with the Golgi apparatus. Dimerization of this enzyme was inhibited by CCCP but did not depend on complex glycosylation of N-linked carbohydrates as assessed by the use of the trimming inhibitor 1-deoxymannojirimycin. It is concluded that dimerization of dipeptidylpeptidase IV occurs in a late Golgi compartment and therefore cannot be a prerequisite for its export from the endoplasmic reticulum.