Unconventional secretory routes: direct protein export across the plasma membrane of mammalian cells

The vast majority of extracellular proteins are exported from mammalian cells by the endoplasmic reticulum/Golgi-dependent secretory pathway. For poorly understood reasons, however, a heterogenous group of extracellular proteins has been discovered that does not make use of signal peptide-dependent secretory transport. Both the release mechanisms and the molecular identity of the secretory machines involved have remained elusive. Recent studies now have established a subgroup of unconventional secretory proteins capable of translocating from the cytoplasm directly across the plasma membrane to get access to the exterior of eukaryotic cells. This review aims to focus on a detailed comparison of the subcellular site of membrane translocation of various unconventional secretory proteins such as the proangiogenic molecule fibroblast growth factor-2 (FGF-2) and Leishmania hydrophilic acylated surface protein B (HASP B). A potential link between membrane translocation and quality control as an integral part of unconventional secretory processes is discussed.     

Lipid translocation across the plasma membrane of mammalian cells.

The plasma membrane, which forms the physical barrier between the intra- and extracellular milieu, plays a pivotal role in the communication of cells with their environment. Exchanging metabolites, transferring signals and providing a platform for the assembly of multi-protein complexes are a few of the major functions of the plasma membrane, each of which requires participation of specific membrane proteins and/or lipids. It is therefore not surprising that the two leaflets of the membrane bilayer each have their specific lipid composition. Although membrane lipid asymmetry has been known for many years, the mechanisms for maintaining or regulating the transbilayer lipid distribution are still not completely understood. Three major players have been presented over the past years: (1) an inward-directed pump specific for phosphatidylserine and phosphatidylethanolamine, known as aminophospholipid translocase; (2) an outward-directed pump referred to as 'floppase' with little selectivity for the polar headgroup of the phospholipid, but whose actual participation in transport of endogenous lipids has not been well established; and (3) a lipid scramblase, which facilitates bi-directional migration across the bilayer of all phospholipid classes, independent of the polar headgroup. Whereas a concerted action of aminophospholipid translocase and floppase could, in principle, account for the maintenance of lipid asymmetry in quiescent cells, activation of the scramblase and concomitant inhibition of the aminophospholipid translocase causes a collapse of lipid asymmetry, manifested by exposure of phosphatidylserine on the cell surface. In this article, each of these transporters will be discussed, and their physiological importance will be illustrated by the Scott syndrome, a bleeding disorder caused by impaired lipid scrambling. Finally, phosphatidylserine exposure during apoptosis will be briefly discussed in relation to inhibition of translocase and simultaneous activation of scramblase.     

Unconventional secretion of fibroblast growth factor 2 and galectin-1 does not require shedding of plasma membrane-derived vesicles

Various molecular mechanisms of unconventional secretion of fibroblast growth factor 2 and galectin-1 have been proposed. A non-vesicular pathway that is based on direct translocation across the plasma membrane has been described. In other studies, however, release into the extracellular space of cell-derived vesicles was implicated in both FGF-2 and Gal-1 secretion. Such vesicles were proposed to originate either from plasma membrane shedding or by the release of exosomes. Employing an inhibitor of plasma membrane blebbing and based on a quantitative biochemical analysis of cell culture supernatants for vesicles potentially carrying FGF-2 or Gal-1, we demonstrate that both FGF-2 and Gal-1 are not exported by shedding of plasma membrane-derived vesicles.     

Unconventional secretion of Acb1 is mediated by autophagosomes

Starving Dictyostelium discoideum cells secrete AcbA, an acyl coenzyme A–binding protein (ACBP) that lacks a conventional signal sequence for entering the endoplasmic reticulum (ER). Secretion of AcbA in D. discoideum requires the Golgi-associated protein GRASP. In this study, we report that starvation-induced secretion of Acb1, the Saccharomyces cerevisiae ACBP orthologue, also requires GRASP (Grh1). This highlights the conserved function of GRASP in unconventional secretion. Although genes required for ER to Golgi or Golgi to cell surface transport are not required for Acb1 secretion in yeast, this process involves autophagy genes and the plasma membrane t-SNARE, Sso1. Inhibiting transport to vacuoles does not affect Acb1 secretion. In sum, our experiments reveal a unique secretory pathway where autophagosomes containing Acb1 evade fusion with the vacuole to prevent cargo degradation. We propose that these autophagosome intermediates fuse with recycling endosomes instead to form multivesicular body carriers that then fuse with the plasma membrane to release cargo.     

Protein translocation across the yeast microsomal membrane is stimulated by a soluble factor

We have found that a soluble activity present in the postribosomal supernatant fraction of Saccharomyces cerevisiae stimulates posttranslational translocation of yeast prepro-alpha-factor across yeast microsomal membranes. Stimulation of translocation is not due to a nonspecific affect on ATP levels. The activity is likely to be due to protein(s) as it is destroyed by N-ethylmaleimide, protease, or heat treatment but not by incubation with RNase. Its apparent sedimentation coefficient is approximately 9.6 S.     

Natural phosphatidylcholine is actively translocated across the plasma membrane to the surface of mammalian cells

The cell surface of eukaryotic cells is enriched in choline phospholipids, whereas the aminophospholipids are concentrated at the cytosolic side of the plasma membrane by the activity of one or more P-type ATPases. Lipid translocation has been investigated mostly by using short chain lipid analogs because assays for endogenous lipids are inherently complicated. In the present paper, we optimized two independent assays for the translocation of natural phosphatidylcholine (PC) to the cell surface based on the hydrolysis of outer leaflet phosphoglycerolipids by exogenous phospholipase A2 and the exchange of outer leaflet PC by a transfer protein. We report that PC reached the cell surface in the absence of vesicular traffic by a pathway that involved translocation across the plasma membrane. In erythrocytes, PC that was labeled at the inside of the plasma membrane was translocated to the cell surface with a half-time of 30 min. This translocation was probably mediated by an ATPase, because it required ATP and was vanadate-sensitive. The inhibition of PC translocation by glibenclamide, an inhibitor of various ATP binding cassette transporters, and its reduction in erythrocytes from both Abcb1a/1b and Abcb4 knockout mice, suggest the involvement of ATP binding cassette transporters in natural PC cell surface translocation. The relative importance of the outward translocation of PC as compared with the well characterized fast inward translocation of phosphatidylserine for the overall asymmetric phospholipid organization in plasma membranes remains to be established.     

Basic fibroblast growth factor stimulates hepatocyte growth factor/scatter factor secretion by human mesenchymal cells

Basic fibroblast growth factor (bFGF) together with other pleiotropic factors plays an important role in many complex physiological processes such as embryonic development, angiogenesis, and wound repair. Among these factors, hepatocyte growth factor/scatter factor (HGF/SF) which is secreted by cells of mesodermal origin exerts its mito- and motogenic activities on cells of epithelial and endothelial origin. Knowledge of the regulatory mechanisms of HGF/SF may contribute to the understanding of its role in physio-pathological processes. We observed that the secretion of HGF/SF by MRC-5 cells and by other fibroblast-derived cell cultures in conditioned media was enhanced by exposure to bFGF. HGF/SF was measured by the scatter assay, a bioassay for cell motility, and was further characterized by Western blot analysis with anti-HGF/SF antibodies. Exposure of MRC-5 cultures to 10 ng/ml of bFGF resulted already 6 h posttreatment in a threefold higher amount of scatter factor secreted into the medium as compared to untreated cultures. HGF/SF secretion was sustained after bFGF treatment for the following 72 h when increased amounts of HGF/SF were detected both in conditioned media as well as associated to the extracellular matrix. The secretion of HGF/SF in cell supernatants increased dose dependently upon treatment with bFGF starting from basal levels of 6 U/ml and reaching 27 U/ml at 30 ng/ml bFGF, plateauing thereafter. Upregulation of HGF/SF by IL-1, already described by others, was confirmed in this study. Based on our findings an articulated interaction can be speculated for bFGF, HGF/SF, and IL-1, e.g., in tissue regeneration during inflammatory processes or in wound healing. © 1996 Wiley-Liss, Inc.     

A direct role for phosphatidylinositol-4,5-bisphosphate in unconventional secretion of fibroblast growth factor 2

Fibroblast growth factor 2 (FGF-2) is a mitogen that is exported from cells by an endoplasmic reticulum/Golgi-independent secretory pathway. Recent findings have shown that FGF-2 export occurs by direct translocation from the cytoplasm across the plasma membrane into the extracellular space. Here, we report that FGF-2 contains a binding site for phosphatidylinositol-4,5-bisphosphate [PI(4,5)P₂], the principal phosphoinositide species associated with plasma membranes. Intriguingly, in the context of a lipid bilayer, the interaction between FGF-2 and PI(4,5)P₂ is shown to depend on a lipid background that resembles plasma membranes. We show that the interaction with PI(4,5)P₂ is critically important for FGF-2 secretion as experimental conditions reducing cellular levels of PI(4,5)P₂ resulted in a substantial drop in FGF-2 export efficiency. Likewise, we have identified FGF-2 variant forms deficient for binding to PI(4,5)P₂ that were found to be severely impaired with regard to export efficiency. These data show that a transient interaction with PI(4,5)P₂ associated with the inner leaflet of plasma membranes represents the initial step of the unconventional secretory pathway of FGF-2.     

Basic fibroblast growth factor is efficiently released from a cytolsolic storage site through plasma membrane disruptions of endothelial cells.

Cells of gut and skin frequently suffer mechanically-induced plasma membrane disruptions in vivo, and bioactive molecules, including basic fibroblast growth factor (bFGF), could enter and leave cytoplasm through these disruptions. We here provide three lines of evidence that bFGF is released with surprising efficiency through plasma membrane disruptions, resembling those known to occur in vivo, produced by scraping endothelial cells from their culturing substratum. First, 41% of the total of bFGF extractable in 1 M NaCl by freeze-thaw and sonication was released simply by scraping the endothelial cells. Second, relative to release of lactate dehydrogenase, cells wounded by scraping under conditions promoting greater than 60% cell survival released a significantly larger amount (up to twofold more) of growth promoting activity than did cells uniformly killed and irreversibly permeabilized by scraping in the cold or by freezing and thawing. Last, cells that survived membrane disruptions released, and contained, less bFGF on each subsequent wounding, consistent with release of bFGF through transient (i.e., survivable) membrane disruptions. A polyclonal antibody against bFGF completely neutralized the growth promoting activity released by scraping, confirming that bFGF is released through endothelial cell plasma membrane disruptions. Cell fractionation and immunolocalization, including a novel permeabilization technique for electron microscope immunolocalization, demonstrated a cytosolic location of bFGF. We conclude that many characteristics of bFGF--its broad spectrum of producing and target cell types, cytosolic location, efficient release through biologically and pathologically relevant plasma membrane wounds, and its release from cells that survive membrane wounds--make it a strong candidate as a "wound hormone" for rapidly initiating the cell growth required for routine maintenance of tissue integrity and/or repair after injury.     

Fibroblast growth factor 9 secretion is mediated by a non-cleaved amino-terminal signal sequence

Fibroblast growth factors are a family of intercellular signaling molecules with multiple and varied roles in animal development. Most are exported from cells by means of a classical amino-terminal signal sequence that is cleaved from the mature protein during its passage through the secretory pathway. Fibroblast growth factor-9 (Fgf-9) does not contain a recognizable signal sequence, although it is efficiently secreted. In this study, we show that Fgf-9 enters the endoplasmic reticulum and traverses the Golgi complex in a similar manner to other constitutively secreted proteins. Deletion and point mutation analysis has revealed an atypical non-cleaved signal sequence within the amino-terminal region of Fgf-9. Moreover, the first 28 amino acids of Fgf-9 can function as an efficient non-cleaved signal peptide when appended to the amino terminus of green fluorescent protein.     

Phloretin-induced changes of lipophilic ion transport across the plasma membrane of mammalian cells.

The adsorption of the hydrophobic anion [W(CO)(5)CN](-) to human lymphoid Jurkat cells gave rise to an additional anti-field peak in the rotational spectra of single cells, indicating that the cell membrane displayed a strong dielectric dispersion in the kilohertz to megahertz frequency range. The surface concentration of the adsorbed anion and its translocation rate constant between the two membrane boundaries could be evaluated from the rotation spectra of cells by applying the previously proposed mobile charge model. Similar single-cell electrorotation experiments were performed to examine the effect of phloretin, a dipolar molecule known to influence the dipole potential of membranes, on the transport of [W(CO)(5)CN](-) across the plasma membrane of mammalian cells. The adsorption of [W(CO)(5)CN](-) was significantly reduced by phloretin, which is in reasonable agreement with the known phloretin-induced effects on artificial and biological membranes. The IC(50) for the effect of phloretin on the transport parameters of the lipophilic ion was approximately 10 microM. The results of this study are consistent with the assumption that the binding of phloretin reduces the intrinsic dipole potential of the plasma membrane. The experimental approach developed here allows the quantification of intrinsic dipole potential changes within the plasma membrane of living cells.     

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.     

Cardioprotection induced by cardiac-specific overexpression of fibroblast growth factor-2 is mediated by the MAPK cascade

Our laboratory showed previously that cardiac-specific overexpression of FGF-2 [FGF-2 transgenic (Tg)] results in increased recovery of contractile function and decreased infarct size after ischemia-reperfusion injury. MAPK signaling is downstream of FGF-2 and has been implicated in other models of cardioprotection. Treatment of FGF-2 Tg and wild-type hearts with U-0126, a MEK-ERK pathway inhibitor, significantly reduced recovery of contractile function after global low-flow ischemia-reperfusion injury in FGF-2 Tg (86 +/- 2% vehicle vs. 66 +/- 4% U-0126; P < 0.05) but not wild-type (61 +/- 7% vehicle vs. 67 +/- 7% U-0126) hearts. Similarly, MEK-ERK inhibition significantly increased myocardial infarct size in FGF-2 Tg (12 +/- 3% vehicle vs. 31 +/- 2% U-0126; P < 0.05) but not wild-type (30 +/- 4% vehicle vs. 36 +/- 7% U-0126) hearts. In contrast, treatment of FGF-2 Tg and wild-type hearts with SB-203580, a p38 inhibitor, did not abrogate FGF-2-induced cardioprotection from postischemic contractile dysfunction. Instead, inhibition of p38 resulted in decreased infarct size in wild-type hearts (30 +/- 4% vehicle vs. 11 +/- 2% SB-203580; P < 0.05) but did not alter infarct size in FGF-2 Tg hearts (12 +/- 3% vehicle vs. 14 +/- 1% SB-203580). Western blot analysis of ERK and p38 activation revealed signaling alterations in FGF-2 Tg and wild-type hearts during early ischemia or reperfusion injury. In addition, MEK-independent ERK inhibition by p38 was observed during early ischemic injury. Together these data suggest that activation of ERK and inhibition of p38 by FGF-2 is cardioprotective during ischemia-reperfusion injury.     

Interaction of platelet factor 4 with fibroblast growth factor 2 is stabilised by heparan sulphate

The CXC chemokine platelet factor 4 (PF4) appears to inhibit tumour growth through its modulation of the activity of angiogenic growth factors. We investigated the heparan sulphate-dependent mechanism of PF4 inhibition of fibroblast growth factor 2 (FGF-2). The ability of PF4 to bind simultaneously to both FGF-2 and HS was assessed using affinity gel chromatography. Thirty-three to forty-two percent more HS bound to the FGF-2 affinity gel in the presence of PF4 than with HS alone. Protection assays showed that PF4 and FGF-2 bound to adjacent or overlapping sites together covering a 12 kDa stretch of HS. This study suggests that the three components may form a ternary complex. PF4 released at sites of angiogenesis may bind to angiogenic growth factors attached to endothelial cell surface HS to disrupt or prevent them from interacting with their signalling receptors. Manipulation of this mechanism may prove useful for clinical intervention of angiogenesis.     

Protein translocation into and across the bacterial plasma membrane and the plant thylakoid membrane

Over the past decade, some familiar themes have emerged on how proteins are inserted into or translocated across the plant chloroplast thylakoid membrane and bacterial inner membranes. In the SecA and signal recognition particle (SRP) pathways, nucleotides and soluble factors are used to translocate proteins across the membrane bilayer in the unfolded state. However, the delta pH-dependent pathway in thylakoids uses a radically different mechanism: transport of proteins across the membrane is driven by the transmembrane pH gradient, and neither stromal factors nor nucleotide triphosphates are needed. In addition, this pathway, which requires the membrane-bound protein Hcf106, appears to translocate proteins in a tightly folded form. Recently, a similar pathway has been shown to operate in eubacteria, and several of its components have been identified.