Identification of rabaptin-5, rabex-5, and GM130 as putative effectors of rab33b, a regulator of retrograde traffic between the Golgi apparatus and ER.

The role of rab33b, a Golgi-specific rab protein, was investigated. Microinjection of rab33b mutants stabilised in the GTP-specific state resulted in a marked inhibition of anterograde transport within the Golgi and in the recycling of glycosyltransferases from the Golgi to the ER, respectively. A GST-rab33b fusion protein stabilised in its GTP form was found to interact by Western blotting or mass spectroscopy with Golgi protein GM130 and rabaptin-5 and rabex-5, two rab effector molecules thought to function exclusively in the endocytic pathway. A similar binding was seen to rab1 but not to rab6, both Golgi rabs. In contrast, rab5 was as expected, shown to bind rabaptin-5 and rabex-5 as well as the endosomal effector protein EEA1 but not GM130. No binding of EEA1 was seen to any of the Golgi rabs.     

Sorting of yeast alpha 1,3 mannosyltransferase is mediated by a lumenal domain interaction, and a transmembrane domain signal that can confer clathrin-dependent Golgi localization to a secreted protein.

alpha 1,3 mannosyltransferase (Mnn1p) is a type II integral membrane protein that is localized to the yeast Golgi complex. We have examined the signals within Mnn1p that mediate Golgi localization by expression of fusion proteins comprised of Mnn1p and the secreted protein invertase. The N-terminal transmembrane domain (TMD) of Mnn1p is sufficient to localize invertase to the Golgi complex by a mechanism that is not saturable by approximately 15-20 fold overexpression. Furthermore, the TMD-mediated localization mechanism is clathrin dependent, as an invertase fusion protein bearing only the Mnn1p TMD is mislocalized to the plasma membrane of a clathrin heavy chain mutant. The Mnn1-invertase fusion proteins are not retained in the Golgi complex as efficiently as Mnn1p, suggesting that other signals may be present in the wild-type protein. Indeed, the Mnn1p lumenal domain (Mnn1-s) is also localized to the Golgi complex when expressed as a functional, soluble protein by exchanging its TMD for a cleavable signal sequence. In contrast to the Mnn1-invertase fusion proteins, overexpression of Mnn1-s saturates its retention mechanism, and results in the partial secretion of this protein. These data indicate that Mnn1p has separable Golgi localization signals within both its transmembrane and lumenal domains.     

Mouse prenylated Rab acceptor is a novel Golgi membrane protein

We have cloned a mouse prenylated Rab acceptor (mPRA), which interacts with various Rab proteins in the yeast two-hybrid system. This study investigated its intracellular localization and characterized the localization signal. The mPRA was found to be an integral membrane protein that was localized to the Golgi complex at steady state as determined by confocal fluorescence microscopy. With green fluorescent protein attached to the N-terminus of mPRA, the fusion protein was expressed in BHK cells and was shown to exhibit the same Golgi localization as the native mPRA. Systematic truncations from the N- and C-termini of mPRA revealed that the entire N-terminal half (91 residues) of the protein was dispensable for the Golgi localization. In contrast, deletion of only 5 residues from the C-terminus diminished the Golgi localization of mPRA, leading to its accumulation in the ER. The data indicate that the C-terminal half (94 residues) of mPRA is necessary and sufficient for proper folding, ER export, and Golgi localization. The Golgi localization of mPRA suggests that it may play a role in the structural organization and function of the Golgi complex.     

Folding and assembly of newly synthesized thyroglobulin occurs in a pre-Golgi compartment

We have investigated the kinetics of folding and dimerization of newly synthesized thyroglobulin (Tg), the precursor protein in the manufacture of thyroid hormone. From the examination of lysates of pulse-labeled cultured thyrocytes by denaturing and nondenaturing gel electrophoresis, we have found that the earliest detectable form of nascent Tg is a transient aggregate, whose dissolution in vitro requires the addition of a reducing agent. In vivo, aggregate dissolution occurs with a t1/2 approximately 10 min at 37 degrees C. By 10 min after synthesis, monomeric Tg is first detectable in a conformationally unstable form. Dimeric Tg is formed thereafter (t1/2 approximately 30 min), but well before arrival of the protein in the medial Golgi (t1/2 approximately 2 h). Certain metabolic inhibitors permit dimerization yet block transport of the dimer to the Golgi. Thus, Tg dimerization occurs in a pre-Golgi compartment, and other steps after dimerization are likely to be important in the process of exit from the endoplasmic reticulum (ER). Further, aggregate dissolution, as well as dimerization, are inhibited significantly at 15 degrees C, indicating thermal sensitivity of Tg folding over and above effects on vesicular transport. Inhibitors of Tg iodination have no effect on Tg dimerization or Golgi arrival. Pretreatment of thyrocytes with thyroid-stimulating hormone substantially accelerates Tg flux through the ER, by increasing the amount, as well as the rate, of Tg transport, possibly at the expense of a small fraction of Tg that appears refractory to dimerization. Inhibition of N-linked glycosylation by tunicamycin causes a complete block in intracellular Tg transport by inducing the formation of biologically irreversible aggregates, suggesting that glycosylation of Tg serves to prevent denaturation of the secretory protein within the ER lumen.     

Protein Kinase A Activity Is Necessary for Fission and Fusion of Golgi to Endoplasmic Reticulum Retrograde Tubules

It is becoming increasingly accepted that together with vesicles, tubules play a major role in the transfer of cargo between different cellular compartments. In contrast to our understanding of the molecular mechanisms of vesicular transport, little is known about tubular transport. How signal transduction molecules regulate these two modes of membrane transport processes is also poorly understood. In this study we investigated whether protein kinase A (PKA) activity regulates the retrograde, tubular transport of Golgi matrix proteins from the Golgi to the endoplasmic reticulum (ER). We found that Golgi-to-ER retrograde transport of the Golgi matrix proteins giantin, GM130, GRASP55, GRASP65, and p115 was impaired in the presence of PKA inhibitors. In addition, we unexpectedly found accumulation of tubules containing both Golgi matrix proteins and resident Golgi transmembrane proteins. These tubules were still attached to the Golgi and were highly dynamic. Our data suggest that both fission and fusion of retrograde tubules are mechanisms regulated by PKA activity.     

The activity of Golgi transport vesicles depends on the presence of the N-ethylmaleimide-sensitive factor (NSF) and a soluble NSF attachment protein (alpha SNAP) during vesicle formation

An assay designed to measure the formation of functional transport vesicles was constructed by modifying a cell-free assay for protein transport between compartments of the Golgi (Balch, W. E., W. G. Dunphy, W. A. Braell, and J. E. Rothman. 1984. Cell. 39:405-416). A 35-kD cytosolic protein that is immunologically and functionally indistinguishable from alpha SNAP (soluble NSF attachment protein) was found to be required during vesicle formation. SNAP, together with the N-ethylmaleimide-sensitive factor (NSF) have previously been implicated in the attachment and/or fusion of vesicles with their target membrane. We show that NSF is also required during the formation of functional vesicles. Strikingly, we found that after vesicle formation, the NEM-sensitive function of NSF was no longer required for transport to proceed through the ensuing steps of vesicle attachment and fusion. In contrast to these functional tests of vesicle formation, SNAP was not required for the morphological appearance of vesicular structures on the Golgi membranes. If SNAP and NSF have a direct role in transport vesicle attachment and/or fusion, as previously suggested, these results indicate that these proteins become incorporated into the vesicle membranes during vesicle formation and are brought to the fusion site on the transport vesicles.     

Reconstitution of brefeldin A-induced golgi tubulation and fusion with the endoplasmic reticulum in semi-intact chinese hamster ovary cells

The fungal metabolite brefeldin A (BFA) induces the disassembly of the Golgi complex in mammalian cells. The drug seems to accentuate tubule formation and causes the subsequent fusion with the endoplasmic reticulum (ER). To investigate the biochemical requirements and kinetics of BFA-induced Golgi disassembly, we have reconstituted the process of green fluorescent protein-tagged Golgi complex disassembly in streptolysin O-permeabilized semi-intact Chinese hamster ovary cells. For quantitative analysis of the morphological changes to the Golgi complex in semi-intact cells, we developed a novel morphometric analysis. Based on this analysis, we have dissected the BFA-induced Golgi disassembly process biochemically into two processes, Golgi tubule formation and fusion with the ER, and found that the formation is induced by only ATP and the residual factors in the cells and that the subsequent fusion is mediated in an N-ethylmaleimide-sensitive factor-dependent manner via Golgi tubules. Tubulation occurs by two pathways that depend on either microtubule integrity or exogenously added cytosol. In the presence of GTPgammaS, coat protein I inhibited the Golgi tubule fusion with the ER but showed no apparent effect on tubulation. Additionally, we analyzed the kinetics of tubulation and fusion independently in nocodazole-treated and -untreated semi-intact cells and found that tubulation is a rate-limiting step of the Golgi disassembly.     

Unexpected intracellular localization of the AMD-associated cystatin C variant

Cystatin C is abundantly expressed by the retinal pigment epithelium (RPE) of the eye. Targeting of cystatin C to the Golgi apparatus and processing through the secretory pathway of RPE cells are dependent upon a 26-amino acid signal sequence of precursor cystatin C. A variant with an alanine (A) to threonine (T) mutation in the penultimate amino acid of the signal sequence (A25T) was recently correlated with increased risk of developing exudative age-related macular degeneration. The biochemical consequence of the A25T mutation upon targeting of the protein is reported here. Targeting and trafficking of full-length mutant (A25T) precursor cystatin C-enhanced green fluorescent protein fusion protein were studied in living, cultured retinal pigment epithelial and HeLa cells. Confocal microscopy studies were substantiated by immunodetection. In striking contrast to wild-type precursor cystatin C fusion protein conspicuously targeted to the Golgi apparatus, the threonine variant was associated principally with mitochondria. Some diffuse fluorescence was also observed throughout the cytoplasm and nucleus (but not nucleoli). Secretion of fusion protein derived from the threonine variant was reduced by approximately 50% compared with that of the wild-type cystatin C fusion protein. Expression of the variant fusion protein did not appear to impair expression or secretion of endogenous cystatin C.     

Stop-and-go movements of plant Golgi stacks are mediated by the acto-myosin system

The Golgi apparatus in plant cells consists of a large number of independent Golgi stack/trans-Golgi network/Golgi matrix units that appear to be randomly distributed throughout the cytoplasm. To study the dynamic behavior of these Golgi units in living plant cells, we have cloned a cDNA from soybean (Glycine max), GmMan1, encoding the resident Golgi protein alpha-1,2 mannosidase I. The predicted protein of approximately 65 kD shows similarity of general structure and sequence (45% identity) to class I animal and fungal alpha-1,2 mannosidases. Expression of a GmMan1::green fluorescent protein fusion construct in tobacco (Nicotiana tabacum) Bright Yellow 2 suspension-cultured cells revealed the presence of several hundred to thousands of fluorescent spots. Immuno-electron microscopy demonstrates that these spots correspond to individual Golgi stacks and that the fusion protein is largely confined to the cis-side of the stacks. In living cells, the stacks carry out stop-and-go movements, oscillating rapidly between directed movement and random "wiggling." Directed movement (maximal velocity 4.2 microm/s) is related to cytoplasmic streaming, occurs along straight trajectories, and is dependent upon intact actin microfilaments and myosin motors, since treatment with cytochalasin D or butanedione monoxime blocks the streaming motion. In contrast, microtubule-disrupting drugs appear to have a small but reproducible stimulatory effect on streaming behavior. We present a model that postulates that the stop-and-go motion of Golgi-trans-Golgi network units is regulated by "stop signals" produced by endoplasmic reticulum export sites and locally expanding cell wall domains to optimize endoplasmic reticulum to Golgi and Golgi to cell wall trafficking.     

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.     

Evidence for a novel route of wheat storage proteins to vacuoles

Wheat seed storage proteins are deposited in protein bodies (PB) inside vacuoles, but their subcellular site of aggregation and their route to vacuoles are still controversial. In the present work, an ultra structural analysis of developing wheat endosperm at early to mid maturation was performed to address these issues. Golgi complexes were rarely detected, indicating that their role in wheat storage protein transport is limited. In contrast, a considerable amount of PB was detected in the cytoplasm. Many of these PB were surrounded by RER membranes and were enlarged by fusion of smaller PB. Small, electron lucent vesicles were detected around the surfaces of the PB in the cytoplasm, or attached to them, suggesting that such attachments and subsequent fusion of the vesicles with each other lead to the formation of small vacuoles containing PB inclusions. Immunogold labeling with serum raised against yeast-BiP, an ER-localized protein, demonstrated that the wheat BiP homolog was present within the PB in the cytoplasm as well as inside vacuoles. This confirmed that the PB were formed within the RER and that the Golgi complex was not involved in their transport to vacuoles. It is concluded that a considerable part of the wheat storage proteins aggregate into PB within the RER and are then transported as intact PB to the vacuoles by a novel route that does not utilize the Golgi complex.     

Asymmetric requirements for a Rab GTPase and SNARE proteins in fusion of COPII vesicles with acceptor membranes

Soluble NSF attachment protein receptor (SNARE) proteins are essential for membrane fusion in transport between the yeast ER and Golgi compartments. Subcellular fractionation experiments demonstrate that the ER/Golgi SNAREs Bos1p, Sec22p, Bet1p, Sed5p, and the Rab protein, Ypt1p, are distributed similarly but localize primarily with Golgi membranes. All of these SNARE proteins are efficiently packaged into COPII vesicles and suggest a dynamic cycling of SNARE machinery between ER and Golgi compartments. Ypt1p is not efficiently packaged into vesicles under these conditions. To determine in which membranes protein function is required, temperature-sensitive alleles of BOS1, BET1, SED5, SLY1, and YPT1 that prevent ER/Golgi transport in vitro at restrictive temperatures were used to selectively inactivate these gene products on vesicles or on Golgi membranes. Vesicles bearing mutations in Bet1p or Bos1p inhibit fusion with wild-type acceptor membranes, but acceptor membranes containing these mutations are fully functional. In contrast, vesicles bearing mutations in Sed5p, Sly1p, or Ypt1p are functional, whereas acceptor membranes containing these mutations block fusion. Thus, this set of SNARE proteins is symmetrically distributed between vesicle and acceptor compartments, but they function asymmetrically such that Bet1p and Bos1p are required on vesicles and Sed5p activity is required on acceptor membranes. We propose the asymmetry in SNARE protein function is maintained by an asymmetric distribution and requirement for the Ypt1p GTPase in this fusion event. When a transmembrane-anchored form of Ypt1p is used to restrict this GTPase to the acceptor compartment, vesicles depleted of Ypt1p remain competent for fusion.     

Intra-Golgi protein transport depends on a cholesterol balance in the lipid membrane

Transport of proteins between intracellular membrane compartments is mediated by a protein machinery that regulates the budding and fusion processes of individual transport steps. Although the core proteins of both processes are defined at great detail, much less is known about the involvement of lipids. Here we report that changing the cellular balance of cholesterol resulted in changes of the morphology of the Golgi apparatus, accompanied by an inhibition of protein transport. By using a well characterized cell-free intra-Golgi transport assay, these observations were further investigated, and it was found that the transport reaction is sensitive to small changes in the cholesterol content of Golgi membranes. Addition as well as removal of cholesterol (10 +/- 6%) to Golgi membranes by use of methyl-beta-cyclodextrin specifically inhibited the intra-Golgi transport assay. Transport inhibition occurred at the fusion step. Modulation of the cholesterol content changed the lipid raft partitioning of phosphatidylcholine and heterotrimeric G proteins, but not of other (non) lipid raft proteins and lipids. We suggest that the cholesterol balance in Golgi membranes plays an essential role in intra-Golgi protein transport and needs to be carefully regulated to maintain the structural and functional organization of the Golgi apparatus.     

Compartmental organization of Golgi-specific protein modification and vacuolar protein sorting events defined in a yeast sec18 (NSF) mutant

The sec18 and sec23 secretory mutants of Saccharomyces cerevisiae have previously been shown to exhibit temperature-conditional defects in protein transport from the ER to the Golgi complex (Novick, P., S. Ferro, and R. Schekman, 1981. Cell. 25:461-469). We have found that the Sec18 and Sec23 protein functions are rapidly inactivated upon shifting mutant cells to the nonpermissive temperature (less than 1 min). This has permitted an analysis of the potential role these SEC gene products play in transport events distal to the ER. The sec-dependent transport of alpha-factor (alpha f) and carboxypeptidase Y (CPY) biosynthetic intermediates present throughout the secretory pathway was monitored in temperature shift experiments. We found that Sec18p/NSF function was required sequentially for protein transport from the ER to the Golgi complex, through multiple Golgi compartments and from the Golgi complex to the cell surface. In contrast, Sec23p function was required in the Golgi complex, but only for transport of alpha f out of an early compartment. Together, these studies define at least three functionally distinct Golgi compartments in yeast. From cis to trans these compartments contain: (a) An alpha 1----6 mannosyltransferase; (b) an alpha 1----3 mannosyltransferase; and (c) the Kex2 endopeptidase. Surprisingly, we also found that a pool of Golgi-modified CPY (p2 CPY) located in a compartment distal to the alpha 1----3 mannosyltransferase does not require Sec18p function for final delivery to the vacuole. This compartment appears to be equivalent to the Kex2 compartment as we show that a novel vacuolar CPY-alpha f-invertase fusion protein undergoes efficient Kex2-dependent cleavage resulting in the secretion of invertase. We propose that this Kex2 compartment is the site in which vacuolar proteins are sorted from proteins destined to be secreted.     

The transmembrane domain of N-glucosaminyltransferase I contains a Golgi retention signal.

The enzyme N-acetylglucosaminyltransferase I (NT, EC 2.4.1.101) is a resident type II transmembrane protein of the Golgi apparatus. To delineate the portion of its primary sequence that is responsible for the Golgi retention of this protein, we constructed chimeras containing different N-terminal portions of NT joined to a reporter sequence, the ectodomain of a type II surface membrane protein. These chimeric proteins were found to be retained in the Golgi apparatus as assessed by cell surface biotinylation and immunofluorescence. We found that the transmembrane domain of NT is sufficient to confer Golgi retention of the fusion proteins and propose that it contains the Golgi retention signal of the parent molecule.     

PRAF1: a Golgi complex transmembrane protein that interacts with viruses.

Prenylated Rab acceptor domain family member 1 (PRAF1), a transmembrane protein whose precise function is unknown, localizes to the Golgi complex, post-Golgi vesicles, lipid rafts, endosomes, and the plasma membrane. VAMP2 and Rab3A are SNARE proteins that interact with PRAF1, and, as part of a SNARE complex, PRAF1 may function in the regulation of docking and fusion of transport vesicles both in the Golgi complex and at the plasma membrane. Alternately, PRAF1 may function as a sorting protein in the Golgi complex. In addition to interacting with SNARE proteins, PRAF1 interacts with rotaviral, retroviral, and herpes viral proteins. The function of viral protein interaction is unknown, but PRAF1 may enhance rotaviral and retroviral assembly. In contrast, PRAF1 may inhibit the herpes virus life cycle.     

VCIP135 deubiquitinase and its binding protein, WAC, in p97ATPase-mediated membrane fusion

Two distinct p97 membrane fusion pathways are required for Golgi biogenesis: the p97/p47 and p97/p37 pathways. VCIP135 is necessary for both pathways, while its deubiquitinating activity is required only for the p97/p47 pathway. We have now identified a novel VCIP135-binding protein, WAC. WAC localizes to the Golgi as well as the nucleus. In Golgi membranes, WAC is involved in a complex containing VCIP135 and p97. WAC directly binds to VCIP135 and increases its deubiquitinating activity. siRNA experiments revealed that WAC is required for Golgi biogenesis. In an in vitro Golgi reformation assay, WAC was necessary only for p97/p47-mediated Golgi reassembly, but not for p97/p37-mediated reassembly. WAC is hence thought to function in p97/p47-mediated Golgi membrane fusion by activating the deubiquitinating function of VCIP135. We also showed that the two p97 pathways function in ER membrane fusion as well. An in vitro ER reformation assay revealed that both pathways required VCIP135 but not its deubiquitinating activity for their ER membrane fusion. This was consistent with the finding that WAC is unnecessary for p97-mediated ER membrane fusion.     

The C2B domain of synaptotagmin I is a Ca2+-binding module

Synaptotagmin I is a synaptic vesicle protein that contains two C(2) domains and acts as a Ca(2+) sensor in neurotransmitter release. The Ca(2+)-binding properties of the synaptotagmin I C(2)A domain have been well characterized, but those of the C(2)B domain are unclear. The C(2)B domain was previously found to pull down synaptotagmin I from brain homogenates in a Ca(2+)-dependent manner, leading to an attractive model whereby Ca(2+)-dependent multimerization of synaptotagmin I via the C(2)B domain participates in fusion pore formation. However, contradictory results have been described in studies of Ca(2+)-dependent C(2)B domain dimerization, as well as in analyses of other C(2)B domain interactions. To shed light on these issues, the C(2)B domain has now been studied using biophysical techniques. The recombinant C(2)B domain expressed as a GST fusion protein and isolated by affinity chromatography contains tightly bound bacterial contaminants despite being electrophoretically pure. The contaminants bind to a polybasic sequence that has been previously implicated in several C(2)B domain interactions, including Ca(2+)-dependent dimerization. NMR experiments show that the pure recombinant C(2)B domain binds Ca(2+) directly but does not dimerize upon Ca(2+) binding. In contrast, a cytoplasmic fragment of native synaptotagmin I from brain homogenates, which includes the C(2)A and C(2)B domains, participates in a high molecular weight complex as a function of Ca(2+). These results show that the recombinant C(2)B domain of synaptotagmin I is a monomeric, autonomously folded Ca(2+)-binding module and suggest that a potential function of synaptotagmin I multimerization in fusion pore formation does not involve a direct interaction between C(2)B domains or requires a posttranslational modification.     

NEX-TRAP, a Novel Method for In Vivo Analysis of Nuclear Export of Proteins

Transport of proteins between cytoplasm and nucleus is mediated by transport factors of the importin α- and β-families and occurs along a gradient of the small GTPase Ran. To date, in vivo analysis as well as prediction of protein nuclear export remain tedious and difficult. We generated a novel bipartite assay called NEX-TRAP (Nuclear EXport Trapped by RAPamycin) for in vivo analysis of protein nuclear export. The assay is based on the rapamycin-induced dimerization of the modules FRB (FK506-rapamycin (FR)-binding domain) and FKBP (FK506-binding protein-12): a potential nuclear export cargo is fused to FRB, to EYFP for direct visualization as well as to an SV40-derived nuclear localization signal (NLS) for constitutive nuclear import. An integral membrane protein that resides at the trans Golgi network (TGN) is fused to a cytoplasmically exposed FKBP and serves as reporter. EYFP-NLS-FRB fusion proteins with export activity accumulate in the nucleus at steady state but continuously shuttle between nucleus and cytoplasm. Rapamycin-induced dimerization of FRB and FKBP at the TGN traps the shuttling protein outside of the nucleus, making nuclear export permanent. Using several example cargoes, we show that the NEX-TRAP is superior to existing assays owing to its ease of use, its sensitivity and accuracy. Analysis of large numbers of export cargoes is facilitated by recombinational cloning. The NEX-TRAP holds the promise of applicability in automated fluorescence imaging for systematic analysis of nuclear export, thereby improving in silico prediction of nuclear export sequences.     

CERAMIDE-GALACTOSYLTRANSFERASE AND CEREBROSIDE-SULPHOTRANSFERASE LOCALISATION IN GOLGI MEMBRANES ISOLATED BY A CONTINUOUS SUCROSE GRADIENT OF MOUSE BRAIN MICROSOMES

Abstract— A 17,000 g supernatant of mouse brain microsomes was subfractionated on a continuous sucrose gradient in order to localise ceramide galactosyltransferase (CGalt, EC 2.4.1.47) and cerebroside sulphotransferase (CST, EC 2.8.2.11), both enzymes involved in the synthesis of myelin lipids. The submicrosomal fractions were analysed for marker enzymes of myelin, plasma membranes, Golgi membranes, endoplasmic reticulum and lysosomes, and their protein distribution was studied. The results and EM studies give evidence that CGalT and CST are located in the Golgi membranes of the brain.