Role of autophagy in unconventional protein secretion

In the secretory pathway, the secretion of proteins to the plasma membrane or to the extracellular milieu occurs via vesicular transport from the endoplasmic reticulum, via the Golgi apparatus, to the plasma membrane. This process and the players involved are understood in considerable detail. However, the mode of secretion of proteins that lack a signal sequence and do not transit through the secretory pathway has not been described, despite the fact that the literature is replete with examples of such proteins. One such protein is an evolutionarily conserved, secreted Acyl-CoA binding protein (known as AcbA in Dictyostelium discoideum, Acb1 in yeast and diazepam-binding inhibitor in mammals). Two recent papers highlighted in this punctum have elucidated the pathways required for the unconventional secretion of Acb1 in Pichia pastoris and Saccharomyces cerevisiae. Both implicate autophagy proteins and autophagosome formation in the process, while also uncovering roles for other interesting proteins in the unconventional secretion of Acb1.     

Role of autophagy in unconventional protein secretion

In the secretory pathway, the secretion of proteins to the plasma membrane or to the extracellular milieu occurs via vesicular transport from the endoplasmic reticulum, via the Golgi apparatus, to the plasma membrane. This process and the players involved are understood in considerable detail. However, the mode of secretion of proteins that lack a signal sequence and do not transit through the secretory pathway has not been described, despite the fact that the literature is replete with examples of such proteins. One such protein is an evolutionarily conserved, secreted Acyl-CoA binding protein (known as AcbA in Dictyostelium discoideum, Acb1 in yeast and diazepam-binding inhibitor in mammals). Two recent papers highlighted in this punctum have elucidated the pathways required for the unconventional secretion of Acb1 in Pichia pastoris and Saccharomyces cerevisiae. Both implicate autophagy proteins and autophagosome formation in the process, while also uncovering roles for other interesting proteins in the unconventional secretion of Acb1.     

dGRASP-mediated noncanonical integrin secretion is required for Drosophila epithelial remodeling

Integral plasma membrane proteins are typically transported in the secretory pathway from the endoplasmic reticulum and the Golgi complex. Here we show that at specific stages of Drosophila development corresponding to morphological changes in epithelia, apposed basolateral membranes separate slightly, allowing new plasma membrane contacts with basal extracellular matrix. At these sites, newly synthesized integrin alpha subunits are deposited via a mechanism that appears to bypass the Golgi. We show that the Drosophila Golgi resident protein dGRASP localizes to these membrane domains and that, in the absence of dGRASP, the integrin subunit is retained intracellularly in both follicular and wing epithelia that are found disrupted. We propose that this dGRASP-mediated noncanonical secretion route allows for developmental regulation of integrin function upon epithelial remodeling. We speculate that this mechanism might be used during development as a means of targeting a specific subset of transmembrane proteins to the plasma membrane.     

The unconventional secretory machinery of fibroblast growth factor 2

Unconventional secretory proteins represent a subpopulation of extracellular factors that are exported from eukaryotic cells by mechanisms that do not depend on the endoplasmic reticulum and the Golgi complex. Various pathways have been implicated in unconventional secretion including those involving intracellular membrane-bound intermediates and others that are based on direct protein translocation across plasma membranes. Interleukin 1β (IL1β) and fibroblast growth factor 2 (FGF2) are classical examples of unconventional secretory proteins with IL1β believed to be present in intracellular vesicles prior to secretion. By contrast, FGF2 represents an example of a non-vesicular mechanism of unconventional secretion. Here, the author discusses the current knowledge about the molecular machinery being involved in FGF2 secretion. To reveal both differential and common requirements, this review further aims at a comprehensive comparison of this mechanism with other unconventional secretory processes. In particular, a potentially general role of tyrosine phosphorylation as a regulatory signal in unconventional protein secretion will be discussed.     

GRASP55 regulates the unconventional secretion and aggregation of mutant huntingtin

Recent studies demonstrated that the Golgi reassembly stacking proteins (GRASPs), especially GRASP55, regulate Golgi-independent unconventional secretion of certain cytosolic and transmembrane cargoes; however, the underlying mechanism remains unknown. Here, we surveyed several neurodegenerative disease–related proteins, including mutant huntingtin (Htt-Q74), superoxide dismutase 1 (SOD1), tau, and TAR DNA–binding protein 43 (TDP-43), for unconventional secretion; our results show that Htt-Q74 is most robustly secreted in a GRASP55-dependent manner. Using Htt-Q74 as a model system, we demonstrate that unconventional secretion of Htt is GRASP55 and autophagy dependent and is enhanced under stress conditions such as starvation and endoplasmic reticulum stress. Mechanistically, we show that GRASP55 facilitates Htt secretion by tethering autophagosomes to lysosomes to promote autophagosome maturation and subsequent lysosome secretion and by stabilizing p23/TMED10, a channel for translocation of cytoplasmic proteins into the lumen of the endoplasmic reticulum–Golgi intermediate compartment. Moreover, we found that GRASP55 levels are upregulated by various stresses to facilitate unconventional secretion, whereas inhibition of Htt-Q74 secretion by GRASP55 KO enhances Htt aggregation and toxicity. Finally, comprehensive secretomic analysis identified novel cytosolic cargoes secreted by the same unconventional pathway, including transgelin (TAGLN), multifunctional protein ADE2 (PAICS), and peroxiredoxin-1 (PRDX1). In conclusion, this study defines the pathway of GRASP55-mediated unconventional protein secretion and provides important insights into the progression of Huntington’s disease.     

GRASP65 and GRASP55 Sequentially Promote the Transport of C-terminal Valine-bearing Cargos to and through the Golgi Complex

The Golgi matrix proteins GRASP65 and GRASP55 have recognized roles in maintaining the architecture of the Golgi complex, in mitotic progression and in unconventional protein secretion whereas, surprisingly, they have been shown to be dispensable for the transport of commonly used reporter cargo proteins along the secretory pathway. However, it is becoming increasingly clear that many trafficking machineries operate in a cargo-specific manner, thus we have investigated whether GRASPs may control the trafficking of selected classes of cargo. We have taken into consideration the C-terminal valine-bearing receptors CD8α and Frizzled4 that we show bind directly to the PSD95-DlgA-zo-1 (PDZ) domains of GRASP65 and GRASP55. We demonstrate that both GRASPs are needed sequentially for the efficient transport to and through the Golgi complex of these receptors, thus highlighting a novel role for the GRASPs in membrane trafficking. Our results open new perspectives for our understanding of the regulation of surface expression of a class of membrane proteins, and suggests the causal mechanisms of a dominant form of autosomal human familial exudative vitreoretinopathy that arises from the Frizzled4 mutation involving its C-terminal valine.     

Phosphatidylserine is a critical modulator for Akt activation

The endoplasmic reticulum (ER)-Golgi-independent, unconventional secretion of Acb1 requires many different proteins. They include proteins necessary for the formation of autophagosomes, proteins necessary for the fusion of membranes with the endosomes, proteins of the multivesicular body pathway, and the cell surface target membrane SNARE Sso1, thereby raising the question of what achieves the connection between these diverse proteins and Acb1 secretion. In the present study, we now report that, upon starvation in Saccharomyces cerevisiae, Grh1 is collected into unique membrane structures near Sec13-containing ER exit sites. Phosphatidylinositol 3 phosphate, the ESCRT (endosomal sorting complex required for transport) protein Vps23, and the autophagy-related proteins Atg8 and Atg9 are recruited to these Grh1-containing membranes, which lack components of the Golgi apparatus and the endosomes, and which we call a novel compartment for unconventional protein secretion (CUPS). We describe the cellular proteins required for the biogenesis of CUPS, which we believe is the sorting station for Acb1's release from the cells.     

Golgi Bypass: Skirting Around the Heart of Classical Secretion

Classical secretion consists of the delivery of transmembrane and soluble proteins to the plasma membrane and the extracellular medium, respectively, and is mediated by the organelles of the secretory pathway, the Endoplasmic Reticulum (ER), the ER exit sites, and the Golgi, as described by the Nobel Prize winner George Palade ( Palade 1975). At the center of this transport route, the Golgi stack has a major role in modifying, processing, sorting, and dispatching newly synthesized proteins to their final destinations. More recently, however, it has become clear that an increasing number of transmembrane proteins reach the plasma membrane unconventionally, either by exiting the ER in non-COPII vesicles or by bypassing the Golgi. Here, we discuss the evidence for Golgi bypass and the possible physiological benefits of it. Intriguingly, at least during Drosophila development, Golgi bypass seems to be mediated by a Golgi protein, dGRASP, which is found ectopically localized to the plasma membrane.The secretion of signal peptide-containing and transmembrane proteins through the cellular organelles that form the secretory pathway has been very well characterized over the years (Rothman 1994; Lee et al. 2004). During their translation, signal peptide-containing proteins are specifically recognized in the cytoplasm by the signal recognition particle and localize to the ER by virtue of the SRP binding its receptor (Nagai et al. 2003; Osborne et al. 2005). Other transmembrane proteins are embedded in the ER membrane by a posttranslational mechanism called C-tail anchoring by the GET complex (Schuldiner et al. 2008). Following transfer into or across the ER membrane, nascent proteins undergo folding, oligomerization, and addition of oligosaccharide chains followed by exit via specialized landmarks, known as ER exit sites (ERES) in mammalian cells and transitional ER (tER) sites in yeast and Drosophila. Both sites are characterized by the presence of cargo-containing coat protein complex II (COPII)-coated vesicles (Bonifacino and Glick 2004; Lee et al. 2004). Thereafter, most proteins are transported through the Golgi (in a manner that is still very much debated) before reaching their final destination, such as the plasma membrane for many transmembrane proteins and the extracellular medium for secreted proteins (Mellman and Warren 2000) (Fig. 1, red arrows).Open in a separate windowFigure 1.Classical trafficking, from the ER to the Golgi to the plasma membrane, is represented by the red arrows. A cargo protein can exit from an ERES in close proximity to the cis-Golgi (route 1a) or a peripheral ERES (route 1b), but irrespective of its ER exit, this protein follows a distinct pathway through the Golgi to the plasma membrane. This pathway is dependent on known SNARE proteins, NSF and SNAPs. As proteins pass from the ER and through the Golgi, their ER-derived high mannose oligosaccharides are modified by addition of complex sugars rendering these proteins EndoH-resistant. BFA treatment or loss of function of intra-Golgi SNAREs would lead to the retention of these proteins in the ER or Golgi and their diminished presence at the plasma membrane.Potential routes for Golgi bypass are represented by blue arrows. Like classical cargo proteins, Golgi bypass cargoes may exit from an ERES near the cis-Golgi (routes 2a,c) or a peripheral ERES (route 2b). However, the immediate fate of these proteins deviates from the classical pathway. A protein following route 2a (from an ERES near the cis-Golgi) or 2b (from a peripheral ERES) would traffic on ER-derived transport intermediates directly to the plasma membrane, routes perhaps taken by CD45 or αPS1. This route would require a specific set of SNAREs, yet to be identified. As these proteins do not pass through the Golgi stack, their high mannose N-glycans remain sensitive to EndoH. These pathways are also revealed by blocking passage through the Golgi either by the application of BFA, or by the loss of function of intra-Golgi SNAREs, (e.g., Syntaxin 5), and observing their continued transport to the plasma membrane. Proteins that follow route 2c would bypass the Golgi stack via an endosomal intermediate, which would facilitate their delivery to the plasma membrane via conventional endosomal fusion machinery. In the case of CFTR, its exit from the ER may occur from either ERES location to the TGN or endosomes. If it is directly delivered to endosomes, it is likely recycled back to the TGN in which the observed oligosaccharide modifications take place before reaching the plasma membrane.More recently, however, several examples of protein trafficking that deviate from this dogma have been discovered. First, an increasing number of cytoplasmic proteins (such as IL-1β, FGF2, MIF, and AcbA/Acb1) that do not harbor a signal peptide are found in the extracellular medium, and these display a wide range of critical activities. This “cytoplasmic protein unconventional secretion” has been extensively discussed elsewhere (Nickel and Seedorf 2008; Nickel and Rabouille 2009) and will not be covered in this volume, except for a brief note toward the end. Second, a small subset of proteins does not exit the ER by virtue of classical COPII-coated vesicles. Third, a few transmembrane proteins have been shown to reach the plasma membrane, bypassing the Golgi, which is the focus of this article.Why some proteins follow an unconventional route of secretion is intriguing but on the whole largely unknown. Through evolution, the cell has segregated processes within membrane compartments to maintain and optimize cellular functions. Why would mechanisms evolve to traffic a subset of proteins via unconventional routes? In this article, we discuss examples of Golgi bypass as well as outline why and how some proteins escape the conventional secretory pathway.     

Insulin-degrading enzyme secretion from astrocytes is mediated by an autophagy-based unconventional secretory pathway in Alzheimer disease

The secretion of proteins that lack a signal sequence to the extracellular milieu is regulated by their transition through the unconventional secretory pathway. IDE (insulin-degrading enzyme) is one of the major proteases of amyloid beta peptide (Aβ), a presumed causative molecule in Alzheimer disease (AD) pathogenesis. IDE acts in the extracellular space despite having no signal sequence, but the underlying mechanism of IDE secretion extracellularly is still unknown. In this study, we found that IDE levels were reduced in the cerebrospinal fluid (CSF) of patients with AD and in pathology-bearing AD-model mice. Since astrocytes are the main cell types for IDE secretion, astrocytes were treated with Aβ. Aβ increased the IDE levels in a time- and concentration-dependent manner. Moreover, IDE secretion was associated with an autophagy-based unconventional secretory pathway, and depended on the activity of RAB8A and GORASP (Golgi reassembly stacking protein). Finally, mice with global haploinsufficiency of an essential autophagy gene, showed decreased IDE levels in the CSF in response to an intracerebroventricular (i.c.v.) injection of Aβ. These results indicate that IDE is secreted from astrocytes through an autophagy-based unconventional secretory pathway in AD conditions, and that the regulation of autophagy is a potential therapeutic target in addressing Aβ pathology.     

Unconventional secretion of fibroblast growth factor 2 is mediated by direct translocation across the plasma membrane of mammalian cells

Fibroblast growth factor 2 (FGF-2) is a pro-angiogenic mediator that is secreted by both normal and neoplastic cells. Intriguingly, FGF-2 has been shown to be exported by an endoplasmic reticulum/Golgi-independent pathway; however, the molecular machinery mediating this process has remained elusive. Here we introduce a novel in vitro system that functionally reconstitutes FGF-2 secretion. Based on affinity-purified plasma membrane inside-out vesicles, we demonstrate post-translational membrane translocation of FGF-2 as shown by protease protection experiments. This process is blocked at low temperature but apparently does not appear to be driven by ATP hydrolysis. FGF-2 membrane translocation occurs in a unidirectional fashion requiring both integral and peripheral membrane proteins. These findings provide direct evidence that FGF-2 secretion is based on its direct translocation across the plasma membrane of mammalian cells. When galectin-1 and macrophage migration inhibitory factor, other proteins exported by unconventional means, were analyzed for translocation into plasma membrane inside-out vesicles, galectin-1 was found to be transported as efficiently as FGF-2. By contrast, migration inhibitory factor failed to traverse the membrane of inside-out vesicles. These findings establish the existence of multiple distinct secretory routes that are operational in the absence of a functional endoplasmic reticulum/Golgi system.     

The Startling Properties of Fibroblast Growth Factor 2: How to Exit Mammalian Cells without a Signal Peptide at Hand

For a long time, protein transport into the extracellular space was believed to strictly depend on signal peptide-mediated translocation into the lumen of the endoplasmic reticulum. More recently, this view has been challenged, and the molecular mechanisms of unconventional secretory processes are beginning to emerge. Here, we focus on unconventional secretion of fibroblast growth factor 2 (FGF2), a secretory mechanism that is based upon direct protein translocation across plasma membranes. Through a combination of genome-wide RNAi screening approaches and biochemical reconstitution experiments, the basic machinery of FGF2 secretion was identified and validated. This includes the integral membrane protein ATP1A1, the phosphoinositide phosphatidylinositol-4,5-bisphosphate (PI(4,5)P₂), and Tec kinase, as well as membrane-proximal heparan sulfate proteoglycans on cell surfaces. Hallmarks of unconventional secretion of FGF2 are: (i) sequential molecular interactions with the inner leaflet along with Tec kinase-dependent tyrosine phosphorylation of FGF2, (ii) PI(4,5)P₂-dependent oligomerization and membrane pore formation, and (iii) extracellular trapping of FGF2 mediated by heparan sulfate proteoglycans on cell surfaces. Here, we discuss new developments regarding this process including the mechanism of FGF2 oligomerization during membrane pore formation, the functional role of ATP1A1 in FGF2 secretion, and the possibility that other proteins secreted by unconventional means make use of a similar mechanism to reach the extracellular space. Furthermore, given the prominent role of extracellular FGF2 in tumor-induced angiogenesis, we will discuss possibilities to develop highly specific inhibitors of FGF2 secretion, a novel approach that may yield lead compounds with a high potential to develop into anti-cancer drugs.     

The molecular mechanism underlying unconventional secretion of Fibroblast Growth Factor 2 from tumour cells

Fibroblast Growth Factor 2 (FGF2) is a potent cell survival factor involved in tumour‐induced angiogenesis. FGF2 is secreted from cells through an unconventional secretory mechanism based upon direct translocation across the plasma membrane. The molecular mechanism underlying this process depends on a surprisingly small set of trans‐acting factors that are physically associated with the plasma membrane. FGF2 membrane translocation is mediated by the ability of FGF2 to oligomerise and to insert into the plasma membrane in a PI(4,5)P₂‐dependent manner. Membrane‐inserted FGF2 oligomers are dynamic translocation intermediates that are disassembled at the extracellular leaflet mediated by membrane proximal heparan sulphate proteoglycans. This process results in the exposure of FGF2 on cell surfaces as part of its unconventional mechanism of secretion. Although the trans‐acting factors and cis‐elements in FGF2 required for unconventional secretion have been known for a while, the core mechanism of this mysterious process has now been reconstituted with purified components establishing the molecular basis of FGF2 secretion from tumour cells.     

Derlin-dependent retrograde transport from endosomes to the Golgi apparatus

Cells have to maintain stable plasma membrane protein and lipid compositions under normal conditions and to remodel their plasma membranes in response to stimuli. This maintenance and remodeling require that integral membrane proteins at the plasma membrane that become misfolded, because of the relatively harsher extracellular milieu or carbohydrate and amino acid sequence changes, are degraded. We had previously shown that Derlin proteins, required for quality control mechanisms in the endoplasmic reticulum, also localize to endosomes and function in the degradation of misfolded integral membrane proteins at the plasma membrane. In this study, we show that Derlin proteins physically associate with sorting nexins that function in retrograde membrane transport from endosomes to the Golgi apparatus. Using genetic studies in Caenorhabditis elegans and ricin pulse-chase analyses in murine RAW264.7 macrophages, we show that the Derlin-sorting nexin interaction is physiologically relevant. Our studies suggest that at least some integral membrane proteins that are misfolded at the plasma membrane are retrogradely transported to the Golgi apparatus and ultimately to the endoplasmic reticulum for degradation via resident quality control mechanisms.     

A region containing a proline-rich motif targets sG(i2) to the golgi apparatus

The central function of heterotrimeric GTP-binding proteins (G proteins) is the transduction of extracellular signals, via membrane receptors, leading to the activation of intracellular effectors. In addition to being associated with the plasma membrane, the alpha subunits of some of these proteins have also been localized in intracellular compartments. The mRNA of the G-protein inhibitory alpha subunit 2 (G(alphai2)) encodes two proteins, G(alphai2) and sG(i2), by an alternative splicing mechanism. sG(i2) differs from G(alphai2) in the C-terminal region and localizes in the Golgi in contrast to the plasma membrane localization of G(alphai2). In this paper we show that the sequence specific to sG(i2) can direct the Golgi localization of other G(alphai) subunits, but not of the stimulatory subunit G(alphas) or of a secreted protein. This indicates that, in addition to the sG(i2) C-terminus, sequences located elsewhere in the protein are required to determine the Golgi localization. Inside the sG(i2) C-terminal region we have identified a 14-amino-acid proline-rich motif which specifies the Golgi localization. Finally, we show that the sG(i2) subunit, once activated, leaves the Golgi to be localized in the endoplasmic reticulum.     

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.     

Myosin VI and its binding partner optineurin are involved in secretory vesicle fusion at the plasma membrane

During constitutive secretion, proteins synthesized at the endoplasmic reticulum (ER) are transported to the Golgi complex for processing and then to the plasma membrane for incorporation or extracellular release. This study uses a unique live-cell constitutive secretion assay to establish roles for the molecular motor myosin VI and its binding partner optineurin in discrete stages of secretion. Small interfering RNA-based knockdown of myosin VI causes an ER-to-Golgi transport delay, suggesting an unexpected function for myosin VI in the early secretory pathway. Depletion of myosin VI or optineurin does not affect the number of vesicles leaving the trans-Golgi network (TGN), indicating that these proteins do not function in TGN vesicle formation. However, myosin VI and optineurin colocalize with secretory vesicles at the plasma membrane. Furthermore, live-cell total internal reflection fluorescence microscopy demonstrates that myosin VI or optineurin depletion reduces the total number of vesicle fusion events at the plasma membrane and increases both the proportion of incomplete fusion events and the number of docked vesicles in this region. These results suggest a novel role for myosin VI and optineurin in regulation of fusion pores formed between secretory vesicles and the plasma membrane during the final stages of secretion.     

Is unconventional secretion inhibited during cell division by Cdk1 activity?

A process of unconventional secretion that is dependent on the Golgi stacking protein GRASP and multiple components of the autophagy machinery has recently been documented for several cytoplasmic and membrane protein. Classical secretion via the exocytic pathway is inhibited during cell division in animal cells, as key membrane compartments, particularly the Golgi, are disassembled and fragmented. The question as to whether unconventional secretion is likewise inhibited during mitosis has not been explored. This mode of secretion supposedly bypasses the Golgi. However, GRASP and Vps34 (a key autophagy protein) are both substrates of the cell cycle regulating cyclin‐dependent kinase 1 (Cdk1), and their activities are apparently inhibited by Cdk1 phosphorylation. Is unconventional secretion therefore similarly inhibited during cell division like conventional secretion? The story may yet turn out to be more complicated. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.     

Golgi and vacuolar membrane proteins reach the vacuole in vps1 mutant yeast cells via the plasma membrane

The Vps1 protein of Saccharomyces cerevisiae is an 80-kD GTPase associated with the Golgi apparatus. Vps1p appears to play a direct role in the retention of late Golgi membrane proteins, which are mislocalized to the vacuolar membrane in its absence. The pathway by which late Golgi and vacuolar membrane proteins reach the vacuole in vps1 delta mutants was investigated by analyzing transport of these proteins in vps1 delta cells that also contained temperature sensitive mutations in either the SEC4 or END4 genes, which are required for a late step in secretion and the internalization step of endocytosis, respectively. Not only was vacuolar transport of a Golgi membrane protein blocked in the vps1 delta sec4-ts and vps1 delta end4-ts double mutant cells at the non-permissive temperature but vacuolar delivery of the vacuolar membrane protein, alkaline phosphatase was also blocked in these cells. Moreover, both proteins expressed in the vps1 delta end4- ts cells at the elevated temperature could be detected on the plasma membrane by a protease digestion assay indicating that these proteins are transported to the vacuole via the plasma membrane in vps1 mutant cells. These data strongly suggest that a loss of Vps1p function causes all membrane traffic departing from the late Golgi normally destined for the prevacuolar compartment to instead be diverted to the plasma membrane. We propose a model in which Vps1p is required for formation of vesicles from the late Golgi apparatus that carry vacuolar and Golgi membrane proteins bound for the prevacuolar compartment.     

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.