S100A13, a member of the S100 gene family of Ca 2؉ -binding proteins has been previously characterized as a component of a brain-derived heparin-binding multiprotein aggregate/complex containing fibroblast growth factor 1 (FGF1). We report that while expression of S100A13 in NIH 3T3 cells results in the constitutive release of S100A13 into the extracellular compartment at 37°C, co-expression of S100A13 with FGF1 represses the constitutive release of S100A13 and enables NIH 3T3 cells to release S100A13 in response to temperature stress. S100A13 release in response to stress occurs with kinetics similar to that observed for the stress-induced release of FGF1, but S100A13 expression is able to reverse the sensitivity of FGF1 release to inhibitors of transcription and translation. The release of FGF1 and S100A13 in response to heat shock results in the solubility of FGF1 at 100% (w/v) ammonium sulfate saturation, and the expression of a S100A13 deletion mutant lacking its novel basic residue-rich domain acts as a dominant negative effector of FGF1 release in vitro. Surprisingly, the expression of S100A13 also results in the stress-induced release of a Cys-free FGF1 mutant, which is normally not released from NIH 3T3 cells in response to heat shock. These data suggest that S100A13 may be a component of the pathway for the release of the signal peptide-less polypeptide, FGF1, and may involve a role for S100A13 in the formation of a noncovalent FGF1 homodimer. FGF11 and FGF2 are the prototype members of a large family of heparin-binding growth factor genes that regulate numerous biological processes such as neurogenesis, mesoderm formation, and angiogenesis (1, 2). FGF1 and FGF2 lack a classical signal peptide sequence that provides access to the conventional endoplasmic reticulum (ER)-Golgi secretion pathway, a characteristic that led to the hypothesis that the release of these polypeptides may proceed through novel release/export pathways (2). Our laboratory previously demonstrated that FGF1, but not FGF2, is released as a latent homodimer by a transcription-and translation-dependent mechanism in response to a variety of cellular stresses including heat shock (3), hypoxia (4), and serum starvation (5). Conversely, the disruption of communication between the ER and Golgi apparatus by brefeldin A does not prevent the release of FGF1 from NIH 3T3 cells, confirming that FGF1 release may occur through a nonconventional pathway (6).FGF1 is released in vitro as a reducing agent-and denaturant-sensitive complex, which contains the p40 extravesicular domain of the Ca 2ϩ -binding protein, p65 synaptotagmin (Syt)1 (7). The release of FGF1 in response to stress is dependent on Syt1 expression, since the expression of either a deletion mutant lacking 95 amino acids from the extravesicular portion of Syt1 or an antisense-Syt1 gene is able to repress FGF1 release in NIH 3T3 cells (7,8). In addition, FGF1 purified from ovine brain as a high molecular weight aggregate exists as a component of a noncovalent heparin-binding complex wit...

Non-classical protein release independent of the ER-Golgi pathway has been reported for an increasing number of proteins lacking an N-terminal signal sequence. The export of FGF1 and IL-1α, two pro-angiogenic polypeptides, provides two such examples. In both cases, export is based on the Cu 2+ -dependent formation of multiprotein complexes containing the S100A13 protein and might involve translocation of the protein across the membrane as a 'molten globule'. FGF1 and IL-1α are involved in pathological processes such as restenosis and tumor formation. Inhibition of their export by Cu 2+ chelators is thus an effective strategy for treatment of several diseases.

Fibroblast growth factor (FGF) 1 is known to be released in response to stress conditions as a component of a multiprotein aggregate containing the p40 extravescicular domain of p65 synaptotagmin (Syt) 1 and S100A13. Since FGF1 is a Cu 2؉ -binding protein and Cu 2؉is known to induce its dimerization, we evaluated the capacity of recombinant FGF1, p40 Syt1, and S100A13 to interact in a cell-free system and the role of Cu 2؉ in this interaction. We report that FGF1, p40 Syt1, and S100A13 are able to bind Cu 2؉ with similar affinity and to interact in the presence of Cu 2؉ to form a multiprotein aggregate which is resistant to low concentrations of SDS and sensitive to reducing conditions and ultracentrifugation. The formation of this aggregate in the presence of Cu 2؉ is dependent on the presence of S100A13 and is mediated by cysteine-independent interactions between S100A13 and either FGF1 or p40 Syt1. Interestingly, S100A13 is also able to interact in the presence of Cu 2؉with Cys-free FGF1 and this observation may account for the ability of S100A13 to export Cys-free FGF1 in response to stress. Lastly, tetrathiomolybdate, a Cu 2؉ chelator, significantly represses in a dose-dependent manner the heat shock-induced release of FGF1 and S100A13. These data suggest that S100A13 may be involved in the assembly of the multiprotein aggregate required for the release of FGF1 and that Cu 2؉ oxidation may be an essential post-translational intracellular modifier of this process. FGF11 and FGF2, the prototype members of a family of heparin-binding growth factors involved in the regulation of neurogenesis, mesoderm formation, and angiogenesis (1, 2), function as extracellular mitogens for diverse populations of target cells, yet they both lack the presence of a classical signal peptide sequence that enables their secretion through the endoplasmic reticulum-Golgi apparatus (1, 2). FGF1, but not FGF2 (3), is released from NIH 3T3 cells in response to heat shock (4), hypoxia (5), and serum starvation (6) in a transcription-and translation-dependent manner, as a biologically inactive homodimer with decreased affinity for heparin (4). Recent studies have suggested that FGF1 may be released as a multiprotein aggregate containing FGF1 and the p40 extravesicular domain of p65 synaptotagmin (Syt)1 (7, 8). These studies are consistent with the observation that FGF1 is present in neuronal tissue as a component of a non-covalent aggregate containing p40 Syt1 and S100A13 (9). It is also known that (i) both p40 Syt1 and S100A13 are constitutively released at 37°C from NIH 3T3 (8, 10), (ii) the expression of FGF1 is able to repress the constitutive release of S100A13 but not p40 Syt1 at 37°C (8, 10), and (iii) FGF1 and S100A13 NIH 3T3 cell cotransfectants are able to release FGF1 and S100A13 in response to temperature stress in a form which alters the solubility of FGF1 at 100% (w/v) ammonium sulfate (10). Moreover, S100A13 appears to play a role in the regulation of FGF1 release since (i) a deletion mutant of S100A13, lacking the carboxyl...