Unconventional secretion of Pichia pastoris Acb1 is dependent on GRASP protein,peroxisomal functions,and autophagosome formation

In contrast to the enormous advances made regarding mechanisms of conventional protein secretion, mechanistic insights into the unconventional secretion of proteins are lacking. Acyl coenzyme A (CoA)–binding protein (ACBP; AcbA in Dictyostelium discoideum), an unconventionally secreted protein, is dependent on Golgi reassembly and stacking protein (GRASP) for its secretion. We discovered, surprisingly, that the secretion, processing, and function of an AcbA-derived peptide, SDF-2, are conserved between the yeast Pichia pastoris and D. discoideum. We show that in yeast, the secretion of SDF-2–like activity is GRASP dependent, triggered by nitrogen starvation, and requires autophagy proteins as well as medium-chain fatty acyl CoA generated by peroxisomes. Additionally, a phospholipase D implicated in soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor–mediated vesicle fusion at the plasma membrane is necessary, but neither peroxisome turnover nor fusion between autophagosomes and the vacuole is essential. Moreover, yeast Acb1 and several proteins required for its secretion are necessary for sporulation in P. pastoris. Our findings implicate currently unknown, evolutionarily conserved pathways in unconventional secretion.     

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.     

Unconventional secretion by autophagosome exocytosis

In this issue, Duran et al. (2010. J. Cell Biol. doi: 10.1083/jcb.200911154) and Manjithaya et al. (2010. J. Cell Biol. doi: 10.1083/jcb.200911149) use yeast genetics to reveal a role for autophagosome intermediates in the unconventional secretion of an acyl coenzyme A (CoA)–binding protein that lacks an endoplasmic reticulum signal sequence. Medium-chain acyl CoAs are also required and may be important for substrate routing to this pathway.In eukaryotic cells, most secreted proteins rely on the highly conserved secretory pathway for their release into the extracellular space. Signal sequences target them for cotranslational translocation across the ER membrane, and the proteins fold within the ER lumen. The proteins are then transported to and through the Golgi apparatus, sorted, and delivered to the cell surface. The machinery responsible for the secretory pathway is comprised of proteins that collect cargo, form transport vesicles, and help vesicles recognize and fuse at the correct target membranes. A small number of secreted proteins use secretory pathway-independent routes by a process called unconventional secretion (Nickel and Rabouille, 2009). In this issue, Duran et al. and Manjithaya et al. make powerful use of yeast genetics to provide new mechanistic insight into the previously unknown, unconventional route taken by an acyl CoA–binding protein (ACBP) to reach the extracellular space.The simplest pathway for unconventional secretion is that taken by the yeast a-factor mating pheromone. This farnesylated and methylated dodecapeptide is exported by the STE6 gene product that encodes an ATP-binding cassette (ABC) family transporter (Kuchler et al., 1989; McGrath and Varshavsky, 1989). Larger proteins, including FGF2, galectins 1 and 3, a subset of interleukins, and the engrailed homeodomain protein are also unconventional secretory cargoes, but their precise routes of export are unknown (Nickel and Rabouille, 2009). During an inflammatory response, interleukin-1β is somehow translocated from the cytosol into secretory lysosomes for release from cells by a still poorly defined mechanism. Caspase-1 may be required for the unconventional secretion of all of these proteins, suggesting that they may use a common route (Keller et al., 2008).Unconventional secretion of an ACBP was first reported in Dictyostelium discoideum. In this organism, spore formation is activated by release of the 10-kD, ER signal sequence lacking, AcbA protein from prespore cells. Secreted AcbA is proteolyzed extracellularly to produce SDF-2 (spore differentiation factor–2; Anjard et al., 1998). Kinseth et al. (2007) showed that a Golgi-associated protein, GRASP, was required for AcbA release and subsequent SDF-2 production but not for cell growth. Inhibitors of ABC family transporters had no influence on AcbA release. Kinseth et al. (2007) revealed an entirely unexpected role for a Golgi protein in unconventional secretion and a route for AcbA that was distinct from a-factor. A conserved role for GRASP was also later reported for the unconventional secretion of α-integrin at a specific stage of Drosophila melanogaster development (Schotman et al., 2008).Duran et al. (2010) now show that secretion of the Saccharomyces cerevisiae AcbA orthologue, Acb1, also requires the corresponding yeast GRASP orthologue, Grh1. As in D. discoideum, nitrogen starvation triggered Acb1 secretion in a concerted pulse. Genes known to be essential for conventional secretion (SEC23, SEC7, or SEC1) or a-factor release (STE6) were not needed for Acb1 release, confirming that the protein uses an unconventional pathway. However, the SEC18 gene that encodes the NSF ATPase was needed. This ATPase disassembles SNARE proteins and is required for all cellular membrane fusion events.The requirement for starvation suggested that an autophagosome intermediate might be involved. Indeed, mutants impaired in various stages of autophagy were all deficient in Acb1 secretion. Fusion of autophagosomes with the vacuole was not required, but the endosomal t-SNARE, Tlg2, and the plasma membrane t-SNARE, Sso1, were required. Finally, convergence of the autophagosomal and the multivesicular endosome pathways was required.Manjithaya et al. (2010) monitored release of Acb1 from the yeast Pichia pastoris by assaying the generation of an SDF-2–like activity that would trigger sporulation in D. discoideum. As in S. cerevisiae, release from P. pastoris required the GRASP homologue Grh1 and numerous autophagy gene products, in particular, Atg11, which is required for receptor-dependent autophagy (Xie and Klionsky, 2007). Similar to the baker’s yeast findings, a plasma membrane t-SNARE was also implicated.Production of medium chain fatty acyl CoAs was needed for Acb1 secretion from P. pastoris. Manjithaya et al. (2010) propose that Acb1 secretion may require that Acb1 bind its medium-chain acyl CoA substrate. Alternatively, the acyl CoA could be needed to acylate a protein (or proteins) that participates in autophagosomal incorporation of Acb1 protein. Lipid modification and/or binding seem to be a recurring theme for unconventional secretion cargoes (Nickel and Rabouille, 2009) and may contribute to incorporation into nascent autophagosomal structures.These experiments suggest that Acb1 is targeted for selective autophagy, a process that begins with recruitment to a so-called phagophore assembly site (Fig. 1). Phagophores are engulfed by multivesicular endosomes that normally deliver their contents to the yeast vacuole (or lysosomes). In some cases, a subset of multivesicular endosomes fuses with the plasma membrane and releases their contents (Simons and Raposo, 2009; Théry et al., 2009). In these studies, fusion of phagophores with multivesicular endosomes and subsequent fusion of these compartments with the plasma membrane appear to represent the major route of unconventional secretion of ACBPs. The use of specific mutant yeast strains has provided key insight into the specific pathways taken by unusual secretory cargoes. These studies also implicate specific SNARE proteins in the poorly understood, multivesicular endosome release process.Open in a separate windowFigure 1.A model for unconventional secretion of Acb1. Selective autophagy involves cargo collection on the surface of a phagophore membrane (blue). These are engulfed by a multivesicular endosome that fuses with the plasma membrane to release its content. Whether the phagophore is released from an endosomal, lumenal vesicle by lipase action before exocytosis (?) is not known. Duran et al. (2010) and Manjithaya et al. (2010) show that the t-SNARE Sso1 is needed for exocytosis, and fusion with the vacuole is not required.What conserved role does GRASP play? A connection between autophagy and the Golgi complex was recently reported by Itoh et al. (2008), who showed a direct link between the autophagy protein Atg16L1 and the Golgi Rab GTPase Rab33b. We do not yet know the precise origins of the phagophore membrane that participates in unconventional secretion, but roles for GRASP and Rab33b suggest that the Golgi is clearly important for this process. Does GRASP help segregate membrane components needed to form a nascent phagophore? How do ACBPs and other unconventionally secreted substrates actually engage the autophagy machinery? ACBP release involves nitrogen starvation; therefore, is stress important for unconventional secretion, and do other stress signals trigger an autophagic response? Important areas for future research include the identification of such signals, the elucidation of the mechanisms by which these signals are translated into cargo sequestration, and determination of the breadth and diversity of proteins that make use of this unconventional secretory pathway.     

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.     

Unconventional Secretion of AcbA in Dictyostelium discoideum through a Vesicular Intermediate

The acyl coenzyme A (CoA) binding protein AcbA is secreted unconventionally and processed into spore differentiation factor 2 (SDF-2), a peptide that coordinates sporulation in Dictyostelium discoideum. We report that AcbA is localized in vesicles that accumulate in the cortex of prespore cells just prior to sporulation. These vesicles are not observed after cells are stimulated to release AcbA but remain visible after stimulation in cells lacking the Golgi reassembly stacking protein (GRASP). Acyl-CoA binding is required for the inclusion of AcbA in these vesicles, and the secretion of AcbA requires N-ethylmaleimide-sensitive factor (NSF). About 1% of the total cellular AcbA can be purified within membrane-bound vesicles. The yield of vesicles decreases dramatically when purified from wild-type cells that were stimulated to release AcbA, whereas the yield from GRASP mutant cells was only modestly altered by stimulation. We suggest that these AcbA-containing vesicles are secretion intermediates and that GRASP functions at a late step leading to the docking/fusion of these vesicles at the cell surface.The acyl coenzyme A (CoA) binding protein (ACBP) has been well characterized for its role in intracellular lipid trafficking, but it also serves as the precursor of peptides that function as intercellular signals. ACBPs are involved in the transport and metabolism of long-chain acyl-CoA esters and steroid biosynthesis (15, 16, 32). In the mammalian brain ACBP is also secreted and processed to generate a diazepam binding inhibitor (DBI) peptide that regulates γ-aminobutyric acid A (GABA_A) ionotropic receptors in neurons (12). Qian and colleagues recently demonstrated the secretion of ACBP from Muller glial cells of the retina (36). In Dictyostelium discoideum the ACBP homolog AcbA is secreted and processed extracellularly into spore differentiation factor 2 (SDF-2), a 34-amino-acid peptide that is highly similar to DBI (2). However, neither ACBP nor AcbA carries a signal sequence that is necessary for entering the endoplasmic reticulum/Golgi pathway. Alternative, unconventional pathways for the secretion of proteins, including ACBPs, have been proposed over the past 20 years, involving the direct membrane transport of proteins, novel membrane trafficking, or autophagy (29, 39).In Dictyostelium, SDF-2 signaling controls the terminal cell differentiation of prespore cells into encapsulated spores during fruiting body formation. Prespore cells within the nascent sorus climb the elongating stalk in a process that requires their active motility (8). The extracellular processing of AcbA into SDF-2 by prestalk cells is thought to coordinate spore encapsulation with fruiting body morphogenesis such that immobile spores are not produced before the stalk begins to form. Approximately halfway through this process of culmination, sporulation occurs as a wave from the top to the bottom of the nascent sorus (38).An understanding of the regulation of SDF-2 signaling is now emerging. During culmination, prespore cells respond to a steroid signal by rapidly releasing GABA, which binds to the GABA_B-like receptor GrlE and stimulates a signal transduction pathway leading to the release of AcbA by prespore cells (3, 5). AcbA is processed into SDF-2 by TagC protease, which is displayed on the surface of prestalk cells in response to GABA. The 34-amino-acid peptide SDF-2 binds to the receptor histidine kinase DhkA, leading to elevated levels of intracellular cyclic AMP (cAMP), which induces spore encapsulation (2, 47). Low levels of SDF-2 also trigger the release of additional AcbA proteins, forming a positive-feedback loop (2, 6). Although only 1 to 3% of the total AcbA is secreted, the levels of SDF-2 in the sorus are far above that required to rapidly induce sporulation (5, 19).The release of AcbA is a critical step in this cascade, but the mechanism of its secretion is largely unknown. The Golgi-associated protein GRASP (Golgi reassembly stacking protein) appears to play an essential role in the process since grpA-null mutants lacking GRASP fail to produce SDF-2 (19). To further explore the role of GRASP and understand the regulation of AcbA secretion, we have determined the subcellular localization of AcbA before and after stimulating its release. Secreted AcbA appears to be localized within membrane-bound vesicles, which accumulate in the cortex of prespore cells during culmination. When AcbA secretion is stimulated by GABA or SDF-2, the cortical vesicles containing AcbA are lost from wild-type cells but remain in cells lacking GRASP. It appears that GRASP is not involved in the production or positioning of AcbA within the cortical vesicles, but it is essential for events leading to their regulated release.     

The Golgi-associated protein GRASP is required for unconventional protein secretion during development

During Dictyostelium development, prespore cells secrete acyl-CoA binding protein (AcbA). Upon release, AcbA is processed to generate a peptide called spore differentiation factor-2 (SDF-2), which triggers terminal differentiation of spore cells. We have found that cells lacking Golgi reassembly stacking protein (GRASP), a protein attached peripherally to the cytoplasmic surface of Golgi membranes, fail to secrete AcbA and, thus, produce inviable spores. Surprisingly, AcbA lacks a signal sequence and is not secreted via the conventional secretory pathway (endoplasmic reticulum-Golgi-cell surface). GRASP is not required for conventional protein secretion, growth, and the viability of vegetative cells. Our findings reveal a physiological role of GRASP and provide a means to understand unconventional secretion and its role in development.     

The multiple facets of the Golgi reassembly stacking proteins

The mammalian GRASPs (Golgi reassembly stacking proteins) GRASP65 and GRASP55 were first discovered more than a decade ago as factors involved in the stacking of Golgi cisternae. Since then, orthologues have been identified in many different organisms and GRASPs have been assigned new roles that may seem disconnected. In vitro, GRASPs have been shown to have the biochemical properties of Golgi stacking factors, but the jury is still out as to whether they act as such in vivo. In mammalian cells, GRASP65 and GRASP55 are required for formation of the Golgi ribbon, a structure which is fragmented in mitosis owing to the phosphorylation of a number of serine and threonine residues situated in its C-terminus. Golgi ribbon unlinking is in turn shown to be part of a mitotic checkpoint. GRASP65 also seems to be the key target of signalling events leading to re-orientation of the Golgi during cell migration and its breakdown during apoptosis. Interestingly, the Golgi ribbon is not a feature of lower eukaryotes, yet a GRASP homologue is present in the genome of Encephalitozoon cuniculi, suggesting they have other roles. GRASPs have no identified function in bulk anterograde protein transport along the secretory pathway, but some cargo-specific trafficking roles for GRASPs have been discovered. Furthermore, GRASP orthologues have recently been shown to mediate the unconventional secretion of the cytoplasmic proteins AcbA/Acb1, in both Dictyostelium discoideum and yeast, and the Golgi bypass of a number of transmembrane proteins during Drosophila development. In the present paper, we review the multiple roles of GRASPs.     

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.     

Phosphatidic acid phospholipase A1 mediates ER–Golgi transit of a family of G protein–coupled receptors

The coat protein II (COPII)–coated vesicular system transports newly synthesized secretory and membrane proteins from the endoplasmic reticulum (ER) to the Golgi complex. Recruitment of cargo into COPII vesicles requires an interaction of COPII proteins either with the cargo molecules directly or with cargo receptors for anterograde trafficking. We show that cytosolic phosphatidic acid phospholipase A1 (PAPLA1) interacts with COPII protein family members and is required for the transport of Rh1 (rhodopsin 1), an N-glycosylated G protein–coupled receptor (GPCR), from the ER to the Golgi complex. In papla1 mutants, in the absence of transport to the Golgi, Rh1 is aberrantly glycosylated and is mislocalized. These defects lead to decreased levels of the protein and decreased sensitivity of the photoreceptors to light. Several GPCRs, including other rhodopsins and Bride of sevenless, are similarly affected. Our findings show that a cytosolic protein is necessary for transit of selective transmembrane receptor cargo by the COPII coat for anterograde trafficking.     

Organization of SNAREs within the Golgi Stack

Antero- and retrograde cargo transport through the Golgi requires a series of membrane fusion events. Fusion occurs at the cis- and trans-side and along the rims of the Golgi stack. Four functional SNARE complexes have been identified mediating lipid bilayer merger in the Golgi. Their function is tightly controlled by a series of reactions involving vesicle tethering and SM proteins. This network of protein interactions spatially and temporally determines the specificity of transport vesicle targeting and fusion within the Golgi.At steady state, the Golgi maintains its structural and functional organization despite a massive lipid and protein flow. A balanced anterograde and retrograde membrane flow are required to constantly recycle the transport machinery and cargo containers (vesicles). In the absence of efficient recycling, directional net cargo transport would cease and the Golgi would collapse. Thus, transport vesicles constantly leave and enter at both sides of the Golgi stack and bud and fuse along the rims of the cisternae. To maintain the compartmental identity, vesicle fusion occurs in a specific and orchestrated manner. These fusion events are mediated by a cascade of reactions centered around the membrane fusion proteins SNAREs (SNAP receptors) (Söllner et al. 1993b).     

COPI-independent Anterograde Transport: Cargo-selective ER to Golgi Protein Transport in Yeast COPI Mutants

The coatomer (COPI) complex mediates Golgi to ER recycling of membrane proteins containing a dilysine retrieval motif. However, COPI was initially characterized as an anterograde-acting coat complex. To investigate the direct and primary role(s) of COPI in ER/Golgi transport and in the secretory pathway in general, we used PCR-based mutagenesis to generate new temperature-conditional mutant alleles of one COPI gene in Saccharomyces cerevisiae, SEC21 (γ-COP). Unexpectedly, all of the new sec21 ts mutants exhibited striking, cargo-selective ER to Golgi transport defects. In these mutants, several proteins (i.e., CPY and α-factor) were completely blocked in the ER at nonpermissive temperature; however, other proteins (i.e., invertase and HSP150) in these and other COPI mutants were secreted normally. Nearly identical cargo-specific ER to Golgi transport defects were also induced by Brefeldin A. In contrast, all proteins tested required COPII (ER to Golgi coat complex), Sec18p (NSF), and Sec22p (v-SNARE) for ER to Golgi transport. Together, these data suggest that COPI plays a critical but indirect role in anterograde transport, perhaps by directing retrieval of transport factors required for packaging of certain cargo into ER to Golgi COPII vesicles. Interestingly, CPY–invertase hybrid proteins, like invertase but unlike CPY, escaped the sec21 ts mutant ER block, suggesting that packaging into COPII vesicles may be mediated by cis-acting sorting determinants in the cargo proteins themselves. These hybrid proteins were efficiently targeted to the vacuole, indicating that COPI is also not directly required for regulated Golgi to vacuole transport. Additionally, the sec21 mutants exhibited early Golgi-specific glycosylation defects and structural aberrations in early but not late Golgi compartments at nonpermissive temperature. Together, these studies demonstrate that although COPI plays an important and most likely direct role both in Golgi–ER retrieval and in maintenance/function of the cis-Golgi, COPI does not appear to be directly required for anterograde transport through the secretory pathway.     

Amino acid permeases require COPII components and the ER resident membrane protein Shr3p for packaging into transport vesicles in vitro

In S. cerevisiae lacking SHR3, amino acid permeases specifically accumulate in membranes of the endoplasmic reticulum (ER) and fail to be transported to the plasma membrane. We examined the requirements of transport of the permeases from the ER to the Golgi in vitro. Addition of soluble COPII components (Sec23/24p, Sec13/31p, and Sar1p) to yeast membrane preparations generated vesicles containing the general amino acid permease. Gap1p, and the histidine permease, Hip1p. Shr3p was required for the packaging of Gap1p and Hip1p but was not itself incorporated into transport vesicles. In contrast, the packaging of the plasma membrane ATPase, Pma1p, and the soluble yeast pheromone precursor, glycosylated pro alpha factor, was independent of Shr3p. In addition, we show that integral membrane and soluble cargo colocalize in transport vesicles, indicating that different types of cargo are not segregated at an early step in secretion. Our data suggest that specific ancillary proteins in the ER membrane recruit subsets of integral membrane protein cargo into COPII transport vesicles.     

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.     

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.     

Secreted Acb1 Contributes to the Yeast-to-Hypha Transition in Cryptococcus neoformans

Adaptation to stress by eukaryotic pathogens is often accompanied by a transition in cellular morphology. The human fungal pathogen Cryptococcus neoformans is known to switch between the yeast and the filamentous form in response to amoebic predation or during mating. As in the classic dimorphic fungal pathogens, the morphotype is associated with the ability of cryptococci to infect various hosts. Many cryptococcal factors and environmental stimuli, including pheromones (small peptides) and nutrient limitation, are known to induce the yeast-to-hypha transition. We recently discovered that secreted matricellular proteins could also act as intercellular signals to promote the yeast-to-hypha transition. Here we show that the secreted acyl coenzyme A (acyl-CoA)-binding protein Acb1 plays an important role in enhancing this morphotype transition. Acb1 does not possess a signal peptide. Its extracellular secretion and, consequently, its function in filamentation are dependent on an unconventional GRASP (Golgi reassembly stacking protein)-dependent secretion pathway. Surprisingly, intracellular recruitment of Acb1 to the secretory vesicles is independent of Grasp. In addition to Acb1, Grasp possibly controls the secretion of other cargos, because the graspΔ mutant, but not the acb1Δ mutant, is defective in capsule production and macrophage phagocytosis. Nonetheless, Acb1 is likely the major or the sole effector of Grasp in terms of filamentation. Furthermore, we found that the key residue of Acb1 for acyl binding, Y80, is critical for the proper subcellular localization and secretion of Acb1 and for cryptococcal morphogenesis.     

Depletion of acyl-coenzyme A-binding protein affects sphingolipid synthesis and causes vesicle accumulation and membrane defects in Saccharomyces cerevisiae

Deletion of the yeast gene ACB1 encoding Acb1p, the yeast homologue of the acyl-CoA-binding protein (ACBP), resulted in a slower growing phenotype that adapted into a faster growing phenotype with a frequency >1:10(5). A conditional knockout strain (Y700pGAL1-ACB1) with the ACB1 gene under control of the GAL1 promoter exhibited an altered acyl-CoA profile with a threefold increase in the relative content of C18:0-CoA, without affecting total acyl-CoA level as previously reported for an adapted acb1Delta strain. Depletion of Acb1p did not affect the general phospholipid pattern, the rate of phospholipid synthesis, or the turnover of individual phospholipid classes, indicating that Acb1p is not required for general glycerolipid synthesis. In contrast, cells depleted for Acb1p showed a dramatically reduced content of C26:0 in total fatty acids and the sphingolipid synthesis was reduced by 50-70%. The reduced incorporation of [(3)H]myo-inositol into sphingolipids was due to a reduced incorporation into inositol-phosphoceramide and mannose-inositol-phosphoceramide only, a pattern that is characteristic for cells with aberrant endoplasmic reticulum to Golgi transport. The plasma membrane of the Acb1p-depleted strain contained increased levels of inositol-phosphoceramide and mannose-inositol-phosphoceramide and lysophospholipids. Acb1p-depleted cells accumulated 50- to 60-nm vesicles and autophagocytotic like bodies and showed strongly perturbed plasma membrane structures. The present results strongly suggest that Acb1p plays an important role in fatty acid elongation and membrane assembly and organization.     

Plant Sar1 isoforms with near-identical protein sequences exhibit different localisations and effects on secretion

In plants, differentiation of subdomains of the endoplasmic reticulum (ER) dedicated to protein export, the ER export sites (ERES), is influenced by the type of export-competent membrane cargo to be delivered to the Golgi. This raises a fundamental biological question: is the formation of transport intermediates at the ER for trafficking to the Golgi always regulated in the same manner? To test this, we followed the distribution and activity of two plant Sar1 isoforms. Sar1 is the small GTPase that regulates assembly of COPII (coat protein complex II) on carriers that transport secretory cargo from ER to Golgi. We show that, in contrast to a tobacco Sar1 isoform, the two Arabidopsis Sar1 GTPases were localised at ERES, independently of co-expression of Golgi-destined membrane cargo in tobacco cells. Although both isoforms labelled ERES, one was found to partition with the membrane fraction to a greater extent. The different distribution of fluorescent fusions of the two isoforms was influenced by the nature of an amino acid residue at the C-terminus of the protein, suggesting that the requirements for membrane association of the two GTPases are not equal. Furthermore, functional analyses based on the secretion of the bulk flow marker α-amylase indicated that over-expression of GTP-restricted mutants of the two isoforms caused different levels of ER export inhibition. These novel results indicate a functional heterogeneity among plant Sar1 isoforms.     

Isoform-specific tethering links the Golgi ribbon to maintain compartmentalization

Homotypic membrane tethering by the Golgi reassembly and stacking proteins (GRASPs) is required for the lateral linkage of mammalian Golgi ministacks into a ribbon-like membrane network. Although GRASP65 and GRASP55 are specifically localized to cis and medial/trans cisternae, respectively, it is unknown whether each GRASP mediates cisternae-specific tethering and whether such specificity is necessary for Golgi compartmentalization. Here each GRASP was tagged with KillerRed (KR), expressed in HeLa cells, and inhibited by 1-min exposure to light. Significantly, inactivation of either GRASP unlinked the Golgi ribbon, and the immediate effect of GRASP65-KR inactivation was a loss of cis- rather than trans-Golgi integrity, whereas inactivation of GRASP55-KR first affected the trans- and not the cis-Golgi. Thus each GRASP appears to play a direct and cisternae-specific role in linking ministacks into a continuous membrane network. To test the consequence of loss of cisternae-specific tethering, we generated Golgi membranes with a single GRASP on all cisternae. Remarkably, the membranes exhibited the full connectivity of wild-type Golgi ribbons but were decompartmentalized and defective in glycan processing. Thus the GRASP isoforms specifically link analogous cisternae to ensure Golgi compartmentalization and proper processing.     

Golgi matrix proteins interact with p24 cargo receptors and aid their efficient retention in the Golgi apparatus.

The Golgi apparatus is a highly complex organelle comprised of a stack of cisternal membranes on the secretory pathway from the ER to the cell surface. This structure is maintained by an exoskeleton or Golgi matrix constructed from a family of coiled-coil proteins, the golgins, and other peripheral membrane components such as GRASP55 and GRASP65. Here we find that TMP21, p24a, and gp25L, members of the p24 cargo receptor family, are present in complexes with GRASP55 and GRASP65 in vivo. GRASPs interact directly with the cytoplasmic domains of specific p24 cargo receptors depending on their oligomeric state, and mutation of the GRASP binding site in the cytoplasmic tail of one of these, p24a, results in it being transported to the cell surface. These results suggest that one function of the Golgi matrix is to aid efficient retention or sequestration of p24 cargo receptors and other membrane proteins in the Golgi apparatus.     

Collagen secretion screening in Drosophila supports a common secretory machinery and multiple Rab requirements

Collagens are large secreted trimeric proteins making up most of the animal extracellular matrix. Secretion of collagen has been a focus of interest for cell biologists in recent years because collagen trimers are too large and rigid to fit into the COPII vesicles mediating transport from the endoplasmic reticulum (ER) to the Golgi. Collagen-specific mechanisms to create enlarged ER-to-Golgi transport carriers have been postulated, including cargo loading by conserved ER exit site (ERES) protein Tango1. Here, we report an RNAi screening for genes involved in collagen secretion in Drosophila. In this screening, we examined distribution of GFP-tagged Collagen IV in live animals and found 88 gene hits for which the knockdown produced intracellular accumulation of Collagen IV in the fat body, the main source of matrix proteins in the larva. Among these hits, only two affected collagen secretion specifically: PH4αEFB and Plod, encoding enzymes known to mediate posttranslational modification of collagen in the ER. Every other intracellular accumulation hit affected general secretion, consistent with the notion that secretion of collagen does not use a specific mode of vesicular transport, but the general secretory pathway. Included in our hits are many known players in the eukaryotic secretory machinery, like COPII and COPI components, SNAREs and Rab-GTPase regulators. Our further analysis of the involvement of Rab-GTPases in secretion shows that Rab1, Rab2 and RabX3, are all required at ERES, each of them differentially affecting ERES morphology. Abolishing activity of all three by Rep knockdown, in contrast, led to uncoupling of ERES and Golgi. We additionally present a characterization of a screening hit we named trabuco (tbc), encoding an ERES-localized TBC domain-containing Rab-GAP. Finally, we discuss the success of our screening in identifying secretory pathway genes in comparison to two previous secretion screenings in Drosophila S2 cells.