Secretion of FGF-16 requires an uncleaved bipartite signal sequence

Fibroblast growth factor (FGF)-16 is one of the rare secreted proteins that do not possess a cleavable signal sequence. Here we describe our examination of the mechanism and structural requirements for the secretion of FGF-16 from COS-1 transfectants. Inhibition of its secretion by brefeldin A and identification of an N-glycan on the secreted form confirmed that FGF-16 is secreted by means of the endoplasmic reticulum and Golgi apparatus, as are secreted proteins having a conventional cleavable signal sequence. Deletion of its N terminus abolished secretion of FGF-16. When chimerized with prolactin, however, the N-terminal sequence of FGF-16 was not able to mediate secretion of the chimera. Point mutations that made the N terminus less hydrophobic had little effect on secretion of FGF-16, whereas making the central hydrophobic region less hydrophobic abolished secretion. Within cells, an unsecretable FGF-16 N-terminal deletion mutant was distributed in the perinuclear region and overlapped the distribution of the Golgi apparatus. Mutants with less hydrophobic central regions were distributed evenly throughout the cytosol. Collectively, these results indicate that FGF-16 employs a unique bipartite signal sequence (i.e. both the N-terminal region and central hydrophobic region) that is not cleaved, although it shares the same secretory machinery used by secreted proteins with cleavable signal sequences.     

Cysteine-rich fibroblast growth factor receptor alters secretion and intracellular routing of fibroblast growth factor 3

Expression of the cysteine-rich fibroblast growth factor (FGF) receptor (CFR) in COS-1 cells strongly inhibits the secretion of co-expressed FGF3. By using a column retention assay and affinity chromatography, we demonstrate that at physiological salt concentrations FGF3 binds with strong affinity to CFR in vivo and in vitro. Furthermore, to show that FGF3 binds to CFR in vivo, truncation mutants of CFR with changed subcellular distributions were shown to cause a similar redistribution of FGF3. Although CFR is a 150-kDa integral membrane glycoprotein that is primarily located in the Golgi apparatus, we show here that in COS-1 cells a substantial proportion of CFR is secreted. This is due to a carboxyl-terminal proteolytic cleavage that releases the intraluminal portion of the protein for secretion. However, the apparent size of the integral membrane and secreted CFR appears similar, since the loss of protein mass is balanced by a gain of complex carbohydrates. The released CFR is associated with the extracellular matrix through its affinity for glycosaminoglycans. These findings show that CFR can modulate the secretion of FGF3 and may control its biological activity by regulating its secretion.     

A hydrophobic region locating at the center of fibroblast growth factor-9 is crucial for its secretion.

Fibroblast growth factor (FGF)-9 is a glycosylated neurotrophic polypeptide highly expressed in brain. The mechanism for its secretion from expressing cells is unclear, because its primary structure lacks a cleavable signal sequence. We, therefore, investigated the mechanism and structural requirements for secretion of FGF-9. As with other secreted proteins, in vitro translation of FGF-9 was inhibited by signal recognition particle, which binds to the signal sequence. When translated in vitro, full-length FGF-9 was translocated into microsomes, glycosylated, and protected from trypsin digestion. By using various FGF-9 deletion mutants, we found that two hydrophobic domains, located at the N terminus and at the center of the FGF-9 primary structure, were crucial for translocation. Examination of various point mutants revealed that local hydrophobicity of the central hydrophobic domain, but not the N terminus, was crucial for translocation. Analogous results were obtained with respect to FGF-9 secretion from transfectant cells. Upon deletion of the complete sequence preceding it, the previously uncleavable hydrophobic domain appeared to serve as a cleavable signal sequence. Our results suggest that nascent FGF-9 polypeptides translocate into endoplasmic reticulum without peptide cleavage via a co-translational pathway in which both the N terminus and the central hydrophobic domain are important; thereafter, FGF-9 is glycosylated and secreted.     

Effects of weakly basic amines on proteolytic processing and terminal glycosylation of secretory proteins in cultured rat hepatocytes.

We examined the effects of weakly basic amines on the secretion and post-translational modifications of secretory proteins in cultured rat hepatocytes. Weakly basic amines such as methylamine, chloroquine and NH4Cl strongly inhibited not only protein secretion, but also the proteolytic conversion of a proform of complement C3, allowing the precursor to be released into the medium. The amines, however, had no effect on the proteolytic conversion of prohaptoglobin into its subunits. Since available evidence indicates that the conversion of pro-C3 occurs at the Golgi complex while that of prohaptoglobin takes place in the endoplasmic reticulum, it is most likely that the weak bases specifically affect the proteolytic event occurring at the Golgi complex. Electron microscopic observations confirmed that the amines caused morphological changes of the Golgi complex, consisting of dilated cisternae and swollen vacuoles. When the glycosylation of alpha 1-protease inhibitor and haptoglobin was examined, it was found that the amines caused a marked accumulation in the cells of both glycoproteins corresponding to the mature secreted forms. Neuraminidase digestion demonstrated that the glycoproteins accumulating in response to the amines had acquired terminal sialic acid. The results indicate that the amines do not significantly affect terminal glycosylation, in contrast with their definite effect on proteolytic processing, despite the fact that both modifications take place in the Golgi complex.     

Secretion of N-glycosylated interleukin-1 beta in Saccharomyces cerevisiae using a leader peptide from Candida albicans. Effect of N-linked glycosylation on biological activity

Human interleukin-1 beta (IL-1 beta) is expressed in activated monocytes as a 31-kDa precursor protein which is processed and secreted as a mature, unglycosylated 17-kDa carboxyl-terminal fragment, despite the fact that it contains a potential N-linked glycosylation site near the NH2 terminus (-Asn7-Cys8-Thr9-). cDNA coding for authentic mature IL-1 beta was fused to the signal sequence from the Candida albicans glucoamylase gene, two amino acids downstream from the signal processing site. Upon expression in Saccharomyces cerevisiae, approximately equimolar amounts of N-glycosylated (22 kDa) and unglycosylated (17 kDa) IL-1 beta protein were secreted. The N-glycosylated yeast recombinant IL-1 beta exhibited a 5-7-fold lower specific activity compared to the unglycosylated species. The mechanism responsible for inefficient glycosylation was also studied. We found no differences in secretion kinetics or processing between the two extracellular forms of IL-1 beta. The 17-kDa protein, which was found to lack core sugars, does not result from deglycosylation of the 22-kDa protein in vivo and does not result from saturation of the glycosylation enzymatic machinery through overexpression. Alteration of the uncommon Cys8 residue in the -Asn-X-Ser/Thr-glycosylation site to Ser also had no effect. However, increasing the distance between Asn7 and the signal processing site increased the extent of core N-linked glycosylation, suggesting a reduction in glycosylation efficiency near the NH2 terminus.     

Secretion of phytohemagglutinin by monkey COS cells

The entire coding region of a gene, which encodes a polypeptide of phytohemagglutinin (PHA-L), obtained from a library of genomic DNA of the common bean Phaseolus vulgaris cv. Greensleeves, was introduced into the SV40 expression vector pJC119. Monkey COS1 cells were transfected with the recombinant clone and the synthesis, glycosylation, and transport of PHA-L studied and compared with the normal processes in bean cotyledons. In the bean, phytohemagglutinin is synthesized on the rough endoplasmic reticulum and transported via the Golgi complex to protein bodies, vacuole-like organelles. Phytohemagglutinin was synthesized and glycosylated at the ER and processed in the Golgi apparatus of the transfected COS1 cells. After passing the Golgi apparatus, PHA-L was slowly secreted into the culture medium (half-time of 3-6 h), a result indicating that the signals for targeting proteins beyond the Golgi apparatus in plant cells are different from those in animal cells. PHA, which is stored in protein bodies in the plant cells, is secreted by animal cells. Tunicamycin inhibited both glycosylation and secretion of PHA by the COS1 cells, a finding indicating an essential role of the oligosaccharides for transport of PHA in these cells in contrast to the situation found in bean cotyledons. PHA, secreted into the culture medium, was partially sensitive to endo H, a result indicating the presence of one high-mannose and one complex oligosaccharide chain, a situation identical to that in beans.     

The rate of bulk flow from the endoplasmic reticulum to the cell surface

Tripeptides containing the acceptor sequence for Asn-linked glycosylation (Asn-X-Ser/Thr) were added to CHO and HepG2 cells. The tripeptides were glycosylated in the ER and then secreted into the medium, via the Golgi complex in which the oligosaccharide chains were processed. The half-time for secretion, approximately 10 min, was faster than that of known proteins transported through the same pathway. Since much evidence suggests that oligosaccharide chains are not signals for transport, it appears that no signal is necessary for rapid and efficient transport from the ER to the Golgi, or from the Golgi to the cell surface. Rather, it appears that proteins retained as permanent residents en route through the ER-Golgi transport pathway must contain specific retention signals.     

FGF3 from Xenopus laevis.

Fibroblast growth factor 3 (FGF3) was first identified as the product of a cellular oncogene activated by mouse mammary tumour virus but its normal role appears to be in the developing embryo. To gain further insights into its function, we have isolated sequences encoding the FGF3 homologue in Xenopus laevis, XFGF3. COS-1 cells transfected with XFGF3 cDNA express a 31 kDa product, p31, generated by signal peptide cleavage and Asn-linked glycosylation at the single consensus site. This product is secreted and becomes associated with the cell surface and extracellular matrix. Proteolytic cleavage of p31 in the extracellular compartment results in an amino-terminally truncated product, p27, that is also glycosylated. Both p31 and p27 bind quantitatively to heparin-Sepharose and can be displaced from the cell surface and extracellular matrix by soluble heparin. Conditioned medium containing these two proteins is capable of inducing transient morphological transformation of NIH3T3 cells and of stimulating DNA synthesis in quiescent C57MG and BALB/MK cells which express different isoforms of FGF receptors 1 and 2. Since XFGF3 behaves very differently from its mouse counterpart, we constructed chimeras in which amino-terminal sequences from XFGF3 were fused with carboxy-terminal sequences from mouse FGF3. Increasing the contribution from mouse FGF3 led to a more restricted host range for the chimeric ligand.     

The N-linked oligosaccharides at the amino terminus of human apoB are important for the assembly and secretion of VLDL

We determined the role of N-linked glycosylation of apolipoprotein B (apoB) in the assembly and secretion of lipoproteins using transfected rat hepatoma McA-RH7777 cells expressing human apoB-17, apoB-37, and apoB-50, three apoB variants with different ability to recruit neutral lipids. Substituting Asn residue with Gln at the single glycosylation site within apoB-17 (N(158)) decreased its secretion efficiency to a level equivalent to that of wild-type apoB-17 treated with tunicamycin, but had little effect on its synthesis or intracellular distribution. When selective N-to-Q substitution was introduced at one or more of the five N-linked glycosylation sites within apoB-37 (N(158), N(956), N(1341), N(1350), and N(1496)), secretion efficiency of apoB-37 from transiently transfected cells was variably affected. When all five N-linked glycosylation sites were mutated within apoB-37, the secretion efficiency and association with lipoproteins were decreased by >50% as compared with wild-type apoB-37. Similarly, mutant apoB-50 with all of its N-linked glycosylation sites mutagenized showed decreased secretion efficiency and decreased lipoprotein association in both d < 1.02 and d > 1.02 g/ml fractions. The inability of mutant apoB-37 and apoB-50 to associate with very low-density lipoproteins was attributable to impaired assembly and was not due to the limitation of lipid availability. The decreased secretion of mutant apoB-17 and apoB-37 was not accompanied by accumulation within the cells, suggesting that the proportion of mutant apoB not secreted was rapidly degraded. However unlike apoB-17 or apoB-37, accumulation of mutant apoB-50 was observed within the endoplasmic reticulum and Golgi compartments. These data imply that the N-glycans at the amino terminus of apoB play an important role in the assembly and secretion of lipoproteins containing the carboxyl terminally truncated apoB.     

A targeting sequence for dense secretory granules resides in the active renin protein moiety of human preprorenin

Human renin plays an important role in blood pressure homeostasis and is secreted in a regulated manner from the juxtaglomerular apparatus of the kidney in response to various physiological stimuli. Many aspects of the regulated release of renin (including accurate processing of prorenin to renin, subcellular targeting of renin to dense secretory granules, and regulated release of active renin) can be reproduced in mouse pituitary AtT-20 cells transfected with a human preprorenin expression vector. Using protein engineering, we have attempted to define the roles of various structures in prorenin that affect its production and trafficking to dense core secretory granules, resulting in its activation and regulated secretion. Replacement of the native signal peptide of human preprorenin with that of a constitutively secreted protein (immunoglobulin M) had no apparent effect on either the constitutive secretion of prorenin or the regulated secretion of active renin in transfected AtT-20 cells. Removal of the pro segment resulted in a marked reduction in total renin secretion, but did not prevent renin from entering the regulated secretory pathway. Single or combined mutations in the two glycosylation sites of human renin did not prevent its regulated secretion; however, the complete elimination of glycosylation resulted in a significant increase in the ratio of renin/prorenin secreted by the transfected cells. Thus, these results suggest that 1) at least one of the sequences that target human renin to dense secretory granules lies within the protein moiety of active renin; 2) the presence of the pro segment is important for efficient prorenin and renin production; and 3) glycosylation can quantitatively affect the proportion of active renin secreted.     

Cleavage of K-FGF produces a truncated molecule with increased biological activity and receptor binding affinity

The K-FGF/HST (FGF-4) growth factor is a member of the FGF family which is efficiently secreted and contains a single N-linked glycosylation signal. To study the role of glycosylation in the secretion of K-FGF, we mutated the human K-fgf cDNA to eliminate the glycosylation signal and the mutated cDNA was cloned into a mammalian expression vector. Studies of immunoprecipitation from the conditioned medium of cells expressing this plasmid revealed that the lack of glycosylation did not impair secretion, however the unglycosylated protein was immediately cleaved into two NH2-terminally truncated peptides of 13 and 15 kD, which appeared to be more biologically active than the wild-type protein. These two proteins also showed higher heparin binding affinity than that of wt K-FGF. We have expressed in bacteria the larger of these two proteins (K140), in which the NH2-terminal 36 amino acids present in the mature form of K-FGF have been deleted. Mitogenicity assays on several cell lines showed that purified recombinant K140 had approximately five times higher biological activity than wild-type recombinant K-FGF. Studies of receptor binding showed that K140 had higher affinity than wt K-FGF for two of the four members of FGF receptor's family, specifically for FGFR-1 (flg) and FGFR-2 (bek). K140 also had increased heparin binding ability, but this property does not appear to be responsible for the increased affinity for FGF receptors. Thus removal of the NH2-terminal 36 amino acids from the mature K-FGF produces growth factor molecules with an altered conformation, resulting in higher heparin affinity, and more efficient binding to FGF receptors. Although it is not clear whether cleavage of K-FGF to generate K140 occurs in vivo, this could represent a novel mechanism of modulation of growth factor activity.     

Secretion of killer toxin encoded on the linear DNA plasmid pGKL1 from Saccharomyces cerevisiae

By the kar1-mediated cytoduction, linear double-stranded DNA plasmids pGKL1 and pGKL2, encoding killer toxin complex, have been successfully transferred to the recipient strains with about 30% frequency. The killer toxin was found to be secreted through the normal yeast secretory pathway by introducing pGKL plasmids into the several Saccharomyces cerevisiae sec mutants and examining the secretion of killer toxin. S. cerevisiae cells, harboring newly isolated deletion plasmid pGKL1D, expressed only the 28K protein among three killer subunits, and secreted the 28K subunit at a level of zero to 20% efficiency of the cells containing intact pGKL1 plasmid. These data indicated that subunit interaction (cosecretion) of killer proteins is required for the efficient secretion of 28K subunit. The 28K precursor protein was found to translocate across the canine pancreatic endoplasmic reticulum membrane under the direction of its own signal peptide in vitro without any other subunits. From kex2 mutant cells harboring pGKL1 plasmid, the 97K subunit, and its precursor 128K protein were not secreted, however, the 28K subunit was secreted in the same amount as that secreted from KEX2 cells. These lines of evidence suggest that the final assembly of killer toxin complex after KEX2 site of Golgi apparatus is not essential for the secretion of 28K subunit, and therefore, that putative interaction between 128K protein and 28K subunit for the transport between endoplasmic reticulum and Golgi apparatus may be required for the efficient secretion of 28K subunit.     

Intracellular regulation of lipoprotein lipase in human monocyte-derived macrophages

The intracellular pathway of lipoprotein lipase (LPL) has been examined in human monocyte-derived macrophages in culture. These cells were previously shown to synthesize and constitutively secrete LPL. The secretion is dependent on new enzyme synthesis. 6-d-old human monocytes have stores of mRNA for linear release of LPL up to 24 h. Enzyme activity in cells and in culture medium was almost completely inhibited by 24 h treatment with tunicamycin, an inhibitor of glycosylation. In monensin-treated cells a pronounced increase in enzyme activity was found, whereas the secreted activity was markedly reduced. This indicates that LPL in human monocytes is processed through a pH sensitive part of the Golgi complex and that the terminal glycosylation is not needed for the expression of its catalytic activity. Our results suggest that lysosomal function is not important in secretion of the enzyme, whereas vesicular transport seem to be involved in regulating LPL in human monocyte-derived macrophages in culture.     

Molecular cloning of a novel cytokine cDNA encoding the ninth member of the fibroblast growth factor family, which has a unique secretion property.

Glia-activating factor (GAF) is a novel heparin-binding growth factor purified from the culture supernatant of a human glioma cell line. It shows a spectrum of activity slightly different from those of other known growth factors. We have isolated the cDNA which encodes human GAF. A homology search revealed that GAF would be the ninth member of the FGF family, and we therefore call it FGF-9. The human FGF-9 cDNA cloned by using oligonucleotide probes encoded a polypeptide consisting of 208 amino acids. Sequence similarity to other members of the FGF family was estimated to be around 30%. Two cysteine residues and other consensus sequences in family members were also well conserved in the FGF-9 sequence. FGF-9 was found to have no typical signal sequence in its N terminus like those in acidic FGF and basic FGF. Acidic FGF and basic FGF are known not to be secreted from cells in a conventional manner. However, FGF-9 was found to be secreted from cells after synthesis despite its lack of a typical signal sequence. It could be detected exclusively in the culture medium of cDNA-transfected COS cells. The amino acid sequence of proteins purified from culture supernatant of the CHO cell line, which was cDNA transfected and selected as a high producer of FGF-9, showed that no peptides were cleaved from the N terminus except the initiation methionine. The rat FGF-9 cDNA was also cloned, and the structural analysis indicated that the PGF-9 gene is highly conserved. Expression of the FGF-9 gene could be detected in the brain and kidney of the adult rat. Restricted gene expression in organs and the unique secretion nature of the protein suggest that FGF-9 plays a physiological role which differs from those of well-characterized acidic FGF and basic FGF.     

Control of the intracellular signaling induced by fibroblast growth factors (FGF) over the proliferation and survival of retinal pigment epithelium cells: example of the signaling regulation of growth factors endogenous to the retina ]

Retinal pigmented epithelium (RPE) cells are of central importance in the maintenance of neural retinal function. RPE cell apoptosis is responsible for the development of a variety of retinal degeneration. The role of FGF2 was investigated on RPE cell proliferation and apoptosis in vitro. In the absence of serum, RPE cells died by apoptosis, while the addition of FGF2 greatly reduces apoptosis over a 7-day culture period. This is due to an autocrine loop involving secretion of endogenous FGF1 in the mechanism that govern FGF2-induced resistance to apoptosis. FGF2 induces long-term activation of FGFR1 and ERK1/2, and production of the anti-apoptotic protein BcL-x. Because FGF1 has no classical signal sequence to direct its secretion, we investigated the effects of FGF1 secretion on RPE proliferation and apoptosis in the absence of exogenous FGF2. Forced secretion of endogenous FGF1 by adding a signal peptide to the FGF1 molecule induces FGF1 secretion and cell proliferation in the presence of serum, while in FGF1 stops to be secreted and cell die in the absence of serum. Conversely, in cells cultured in the presence of serum, FGF1 without signal peptide is not secreted, but is secreted and rescue RPE cell from apoptosis when cells are cultured without serum. Thus, the proliferation and survival activities of endogenous FGF1 depend on the secretion of FGF1 which is determined by the cell environment.     

Maturation of lipoprotein lipase. Expression of full catalytic activity requires glucose trimming but not translocation to the cis-Golgi compartment.

The relationship between maturation of lipoprotein lipase (LPL) and its translocation from the endoplasmic reticulum (ER) to the Golgi complex was determined by measuring lipolytic activity under conditions preventing transport of the enzyme from the ER to the Golgi compartment. In the presence of brefeldin A, a reagent that inhibits movement of proteins from the ER and causes the disassembly of the Golgi complex, pro-5 Chinese hamster ovary cells accumulated catalytically active LPL, while secretion of the enzyme was effectively blocked. LPL retained intracellularly by brefeldin A treatment possessed oligosaccharide chains that were processed to the complex form by the Golgi enzymes redistributed into the ER. At 16 degrees C, a condition disrupting protein transport to the cis-Golgi, the retained enzyme again remained catalytically active although the oligosaccharides remained in the high mannose form. Lastly, attachment of the specific ER retention signal KDEL (Lys-Asp-Glu-Leu) to the carboxyl terminus of LPL also resulted in intracellularly retained enzyme that was fully active. The importance of oligosaccharide processing for attainment of LPL catalytic activity in vitro was also determined. LPL was active and secreted when trimming of the mannose residues was inhibited by deoxymannojirimycin and when addition of complex sugars was blocked using Chinese hamster ovary mutants (lec1 and lec2), indicating that these processing events are not necessary for the expression of a functional enzyme. However, blocking glucose removal by glucosidase inhibitors (castanospermine and N-methyl-deoxynojirimycin) resulted in a significant reduction in LPL specific activity and secretion. Thus, glucose trimming of LPL oligosaccharides is essential for enzyme activation; however, further oligosaccharide processing or translocation of the enzyme to the cis-Golgi is not required for full expression of lipolytic activity in vitro.     

Protein folding does not prevent the nonclassical export of FGF1 and S100A13

Newly synthesized proteins are usually exported through the endoplasmic reticulum (ER) and Golgi due to the presence in their primary sequence of a hydrophobic signal peptide that is recognized by the ER translocation system. However, some secreted proteins lack a signal peptide and are exported independently of ER-Golgi. Fibroblast growth factor (FGF)1 is included in this group of polypeptides, as well as S100A13 that is a small calcium-binding protein critical for FGF1 export. Classically secreted proteins are transported into ER in their unfolded states. To determine the role of protein tertiary structure in FGF1 export through the cell membrane, we produced the chimeras of FGF1 and S100A13 with dihydrofolate reductase (DHFR). The specific DHFR inhibitor, aminopterin, prevents its unfolding. We found that aminopterin did not inhibit the release of FGF1:DHFR and S100A13:DHFR. Thus, FGF1 and S100A13 can be exported in folded conformation.     

Tyrosine sulfation is not the last modification of entactin before its secretion from 3T3-L1 adipocytes

Tyrosine sulfation of entactin was studied by labeling of 3T3-L1 adipocytes with [35S]methionine or H2 35SO4 in the presence or absence of tunicamycin or monensin. Four precursors (EN1-4) at different steps of modification were detected in addition to mature entactin. Under normal conditions, EN2 and mature entactin were intracellular species, and the latter was sulfated and secreted. Inhibition of co-translational transfer of N-linked oligosaccharides by tunicamycin produced EN1 and EN3 as intracellular species, and EN3 was sulfated and secreted. Interruption of protein transport from medial to trans (distal) Golgi cisternae by monensin, and consequent blockage of terminal glycosylation caused intracellular accumulation of EN4. EN4 was sulfated and of different size compared to mature entactin. These facts suggested that tyrosine sulfation of entactin occurs in medial Golgi cisternae and is not the last modification before its secretion. Our results appeared inconsistent with recent observations by Baeuerle and Huttner [(1987) J. Cell Biol. 105, 2655-2664] that tyrosine sulfation of IgM occurred within the trans (distal) Golgi cisternae as the last modification before its exit from the Golgi complex.     

Endogenous tagging reveals a mid-Golgi localization of the glycosyltransferase-cleaving intramembrane protease SPPL3

Numerous Golgi-resident enzymes implicated in glycosylation are regulated by the conserved intramembrane protease SPPL3. SPPL3-catalyzed endoproteolysis separates Golgi enzymes from their membrane anchors, enabling subsequent release from the Golgi and secretion. Experimentally altered SPPL3 expression changes glycosylation patterns, yet the regulation of SPPL3-mediated Golgi enzyme cleavage is not understood and conflicting results regarding the subcellular localization of SPPL3 have been reported. Here, we used precise genome editing to generate isogenic cell lines expressing N- or C-terminally tagged SPPL3 from its endogenous locus. Using these cells, we conducted co-localization analyses of tagged endogenous SPPL3 and Golgi markers under steady-state conditions and upon treatment with drugs disrupting Golgi organization. Our data demonstrate that endogenous SPPL3 is Golgi-resident and found predominantly in the mid-Golgi. We find that endogenous SPPL3 co-localizes with its substrates but similarly with non-substrate type II proteins, demonstrating that in addition to co-localization in the Golgi other substrate-intrinsic properties govern SPPL3-mediated intramembrane proteolysis. Given the prevalence of SPPL3-mediated cleavage among Golgi-resident proteins our results have important implications for the regulation of SPPL3 and its role in the organization of the Golgi glycosylation machinery.     

Processing, secretion, and biological properties of a novel growth factor of the fibroblast growth factor family with oncogenic potential.

We recently reported that the protein encoded in a novel human oncogene isolated from Kaposi sarcoma DNA was a growth factor with significant homology to basic and acidic fibroblast growth factors (FGFs). To study the properties of this growth factor (referred to as K-FGF) and the mechanism by which the K-fgf oncogene transforms cells, we have studied the production and processing of K-FGF in COS-1 cells transfected with a plasmid encoding the K-fgf cDNA. The results show that, unlike basic and acidic FGFs, the K-FGF protein is cleaved after a signal peptide, glycosylated, and efficiently secreted as a mature protein of 176 or 175 amino acids. Inhibition of glycosylation impaired secretion, and the stability of the secreted K-FGF was greatly enhanced by the presence of heparin in the cultured medium. We have used the conditioned medium from transfected COS-1 cells to test K-FGF biological activity. Similar to basic FGF, the K-FGF protein was mitogenic for fibroblasts and endothelial cells and induced the growth of NIH 3T3 mouse cells in serum-free medium. Accordingly, K-fgf-transformed NIH 3T3 cells grew in serum-free medium, consistent with an autocrine mechanism of growth. We have also expressed the protein encoded in the K-fgf protooncogene in COS-1 cells, and it was indistinguishable in its molecular weight, glycosylation, secretion, and biological activity from K-FGF. Taken together, these results suggest that the mechanism of activation of this oncogene is due to overexpression rather than to mutations in the coding sequences.