The germinal centre kinase Don3 is crucial for unconventional secretion of chitinase Cts1 in Ustilago maydis

Unconventional secretion has emerged as an increasingly important cellular process in eukaryotic cells. The underlying translocation mechanisms are diverse and often little understood. We study unconventional secretion of chitinase Cts1 in the corn smut fungus Ustilago maydis. This protein participates in the cytokinesis of yeast cells. During budding it localizes to the septated fragmentation zone where it presumably functions in the degradation of remnant chitin to allow separation of mother and daughter cell. However, the mechanistic details of Cts1 export remain unclear.Here we investigated the mechanism of unconventional Cts1 secretion with a focus on cytokinesis. Cell-cycle inhibition experiments supported the hypothesis that Cts1 export is connected to cytokinesis. To substantiate this finding we analysed gene deletion mutants impaired in cell separation and discovered that strains defective in secondary septum formation were affected in Cts1 export. The germinal centre kinase Don3 had a particularly strong influence on unconventional secretion. Using a synthetic switch, we unambiguously verified an essential role of Don3 for cytokinesis-dependent Cts1 export via the fragmentation zone. Thus, we gained novel insights into the mechanism of unconventional secretion and discovered the first regulatory component of this process.     

Rerouting of fibroblast growth factor 2 to the classical secretory pathway results in post-translational modifications that block binding to heparan sulfate proteoglycans

FGF-2 is a proangiogenic growth factor secreted by unconventional means. It is unknown why FGF-2 takes an ER/Golgi-independent secretory route. We find that secretion of FGF-2 via the ER/Golgi system causes post-translational modifications that prevent binding to heparan sulfate proteoglycans (HSPGs), an interaction that is critically important for both FGF-2 storage and signal transduction. This loss of function is due to artificial O-glycosylation mainly resulting in the addition of glycosaminoglycan chains of the chrondroitin sulfate type. Our findings suggest that the unconventional mechanism of FGF-2 export is an ancient pathway of protein secretion that, in the course of evolution, has been kept due to the inability of the classical secretory pathway to export FGF-2 in a functional form.     

Pitfalls and opportunities in the characterization of unconventionally secreted proteins by secretome analysis

Proteins are released from cells by different secretory pathways. The secretory pathway via the ER-Golgi route - realized by a signal sequence - is referred to as “classical secretion”. In contrast, alternative secretory pathways were summarized as “unconventional protein secretion”. Until now, unconventional protein secretion was lacking attention due to the absence of detailed mechanistic insight and limited experimental access. However, there is a growing number of experimental data showing that a large proportion of secreted proteins is released by these alternative routes. Secretomics - the analysis of all secreted proteins of a cell population - offers the opportunity to gain more functional insight into unconventional protein secretion. Several pitfalls in secretome analysis starting with the analyzed cell model and sample preparation to data analysis have to be considered for detailed characterization of the secretome. Here, we highlight the investigation of secretomes by quantitative LC-MS/MS analysis and discuss pitfalls and opportunities in the characterization of unconventionally secreted proteins by secretome analysis.     

Autophagy-based unconventional secretory pathway for extracellular delivery of IL-1β

Autophagy controls the quality and quantity of the eukaryotic cytoplasm while performing two evolutionarily highly conserved functions: cell-autonomous provision of energy and nutrients by cytosol autodigestion during starvation, and removal of defunct organelles and large aggregates exceeding the capacity of other cellular degradative systems. In contrast to these autodigestive processes, autophagy in yeast has additional, biogenesis functions. However, no equivalent biosynthetic roles have been described for autophagy in mammals. Here, we show that in mammalian cells, autophagy has a hitherto unappreciated positive contribution to the biogenesis and secretion of the proinflammatory cytokine IL-1β via an export pathway that depends on Atg5, inflammasome, at least one of the two mammalian Golgi reassembly stacking protein (GRASP) paralogues, GRASP55 (GORASP2) and Rab8a. This process, which is a type of unconventional secretion, expands the functional manifestations of autophagy beyond autodigestive and quality control roles in mammals. It enables a subset of cytosolic proteins devoid of signal peptide sequences, and thus unable to access the conventional pathway through the ER, to enter an autophagy-based secretory pathway facilitating their exit from the cytoplasm.     

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.     

Exploration of twin‐arginine translocation for expression and purification of correctly folded proteins in Escherichia coli

Historically, the general secretory (Sec) pathway of Gram‐negative bacteria has served as the primary route by which heterologous proteins are delivered to the periplasm in numerous expression and engineering applications. Here we have systematically examined the twin‐arginine translocation (Tat) pathway as an alternative, and possibly advantageous, secretion pathway for heterologous proteins. Overall, we found that: (i) export efficiency and periplasmic yield of a model substrate were affected by the composition of the Tat signal peptide, (ii) Tat substrates were correctly processed at their N‐termini upon reaching the periplasm and (iii) proteins fused to maltose‐binding protein (MBP) were reliably exported by the Tat system, but only when correctly folded; aberrantly folded MBP fusions were excluded by the Tat pathway's folding quality control feature. We also observed that Tat export yield was comparable to Sec for relatively small, well‐folded proteins, higher relative to Sec for proteins that required cytoplasmic folding, and lower relative to Sec for larger, soluble fusion proteins. Interestingly, the specific activity of material purified from the periplasm was higher for certain Tat substrates relative to their Sec counterparts, suggesting that Tat expression can give rise to relatively pure and highly active proteins in one step.     

The mystery of nonclassical protein secretion. A current view on cargo proteins and potential export routes.

Most of the examples of protein translocation across a membrane (such as the import of classical secretory proteins into the endoplasmic reticulum, import of proteins into mitochondria and peroxisomes, as well as protein import into and export from the nucleus), are understood in great detail. In striking contrast, the phenomenon of unconventional protein secretion (also known as nonclassical protein export or ER/Golgi-independent protein secretion) from eukaryotic cells was discovered more than 10 years ago and yet the molecular mechanism and the molecular identity of machinery components that mediate this process remain elusive. This problem appears to be even more complex as several lines of evidence indicate that various kinds of mechanistically distinct nonclassical export routes may exist. In most cases these secretory mechanisms are gated in a tightly controlled fashion. This review aims to provide a comprehensive overview of our current knowledge as a basis for the development of new experimental strategies designed to unravel the molecular machineries mediating ER/Golgi-independent protein secretion. Beyond solving a fundamental problem in current cell biology, the molecular analysis of these processes is of major biomedical importance as these export routes are taken by proteins such as angiogenic growth factors, inflammatory cytokines, components of the extracellular matrix which regulate cell differentiation, proliferation and apoptosis, viral proteins, and parasite surface proteins potentially involved in host infection.     

Type III export: new uses for an old pathway

Gram-negative bacteria use type III secretion (TTS) systems to translocate proteins into the extracellular environment or directly into eukaryotic cells. These complex secretory systems are assembled from over 20 different structural proteins, including 10 that have counterparts in the flagellar export pathway. Secretion substrates are directed to the TTS machinery via mRNA and/or amino acid secretion signals. TTS chaperones bind to select secretion substrates and assist in the export process. Recent progress in the understanding of TTS is reviewed.     

Cysteineless non-glycosylated monomeric blue fluorescent protein,secBFP2, for studies in the eukaryotic secretory pathway

Fluorescent protein (FP) technologies suitable for use within the eukaryotic secretory pathway are essential for live cell and protein dynamic studies. Localization of FPs within the endoplasmic reticulum (ER) lumen has potentially significant consequences for FP function. All FPs are resident cytoplasmic proteins and have rarely been evolved for the chemically distinct environment of the ER lumen. In contrast to the cytoplasm, the ER lumen is oxidizing and the site where secretory proteins are post-translationally modified by disulfide bond formation and N-glycosylation on select asparagine residues. Cysteine residues and N-linked glycosylation consensus sequences were identified within many commonly utilized FPs. Here, we report mTagBFP is post-translationally modified when localized to the ER lumen. Our findings suggest these modifications can grossly affect the sensitivity and reliability of FP tools within the secretory pathway. To optimize tools for studying events in this important intracellular environment, we modified mTagBFP by mutating its cysteines and consensus N-glycosylation sites. We report successful creation of a secretory pathway-optimized blue FP, secBFP2.     

The RNA-binding protein Rrm4 is essential for efficient secretion of endochitinase Cts1

Long-distance transport of mRNAs is crucial in determining spatio-temporal gene expression in eukaryotes. The RNA-binding protein Rrm4 constitutes a key component of microtubule-dependent mRNA transport in filaments of Ustilago maydis. Although a number of potential target mRNAs could be identified, cellular processes that depend on Rrm4-mediated transport remain largely unknown. Here, we used differential proteomics to show that ribosomal, mitochondrial, and cell wall-remodeling proteins, including the bacterial-type endochitinase Cts1, are differentially regulated in rrm4Δ filaments. In vivo UV crosslinking and immunoprecipitation and fluorescence in situ hybridization revealed that cts1 mRNA represents a direct target of Rrm4. Filaments of cts1Δ mutants aggregate in liquid culture suggesting an altered cell surface. In wild type cells Cts1 localizes predominantly at the growth cone, whereas it accumulates at both poles in rrm4Δ filaments. The endochitinase is secreted and associates most likely with the cell wall of filaments. Secretion is drastically impaired in filaments lacking Rrm4 or conventional kinesin Kin1 as well as in filaments with disrupted microtubules. Thus, Rrm4-mediated mRNA transport appears to be essential for efficient export of active Cts1, uncovering a novel molecular link between mRNA transport and the mechanism of secretion.     

Secretion of nuclease across the outer membrane of Serratia marcescens and its energy requirements.

Extracellular secretion of Serratia marcescens nuclease occurs as a two-step process via a periplasmic intermediate. Unlike other extracellular proteins secreted by gram-negative bacteria by the general secretory pathway, nuclease accumulates in the periplasm in its active form for an unusually long time before its export into the growth medium. The energy requirements for extracellular secretion of nuclease from the periplasm were investigated. Our results suggest that the second step of secretion across the outer membrane is dependent upon the external pH; acidic pH effectively but reversibly blocks extracellular secretion. However, electrochemical proton gradient, and possibly ATP hydrolysis, are not required for this step. We suggest that nuclease uses a novel mechanism for the second step of secretion in S. marcescens.     

Resting (basal) secretion of proteins is provided by the minor regulated and constitutive-like pathways and not granule exocytosis in parotid acinar cells

Resting secretion of salivary proteins by the parotid gland is sustained in situ between periods of eating by parasympathetic stimulation and has been assumed to involve low level granule exocytosis. By using parotid lobules from ad libitum fed rats stimulated with low doses of carbachol as an in vitro analog of resting secretion, we deduce from the composition of discharged proteins that secretion does not involve granule exocytosis. Rather, it derives from two other acinar export routes, the constitutive-like (stimulus-independent) pathway and the minor regulated pathway, which responds to low doses of cholinergic or beta-adrenergic agonists (Castle, J. D., and Castle, A. M. (1996) J. Cell Sci. 109, 2591-2599). The protein composition collected in vitro mimics that collected from cannulated ducts of glands given low level stimulation in situ. Analysis of secretory trafficking along the two pathways of resting secretion has indicated that the constitutive-like pathway may pass through endosomes after diverging from the minor regulated pathway at a brefeldin A-sensitive branch point. The branch point is deduced to be distal to a common vesicular budding event by which both pathways originate from immature granules. Detectable perturbation of neither pathway in lobules was observed by wortmannin addition, and neither serves as a significant export route for lysosomal procathepsin B. These findings show that parotid acinar cells use low capacity, high sensitivity secretory pathways for resting secretion and reserve granule exocytosis, a high capacity, low sensitivity pathway, for massive salivary protein export during meals. An analogous strategy may be employed in other secretory cell types.     

Unconventional secretion of tissue transglutaminase involves phospholipid-dependent delivery into recycling endosomes

Although endosomal compartments have been suggested to play a role in unconventional protein secretion, there is scarce experimental evidence for such involvement. Here we report that recycling endosomes are essential for externalization of cytoplasmic secretory protein tissue transglutaminase (tTG). The de novo synthesized cytoplasmic tTG does not follow the classical ER/Golgi-dependent secretion pathway, but is targeted to perinuclear recycling endosomes, and is delivered inside these vesicles prior to externalization. On its route to the cell surface tTG interacts with internalized β1 integrins inside the recycling endosomes and is secreted as a complex with recycled β1 integrins. Inactivation of recycling endosomes, blocking endosome fusion with the plasma membrane, or downregulation of Rab11 GTPase that controls outbound trafficking of perinuclear recycling endosomes, all abrogate tTG secretion. The initial recruitment of cytoplasmic tTG to recycling endosomes and subsequent externalization depend on its binding to phosphoinositides on endosomal membranes. These findings begin to unravel the unconventional mechanism of tTG secretion which utilizes the long loop of endosomal recycling pathway and indicate involvement of endosomal trafficking in non-classical protein secretion.     

Secretion of the galectin family of mammalian carbohydrate-binding proteins

Galectins are cytosolic proteins that lack any signal sequence for transport into the endoplasmic reticulum and are not glycosylated, although several galectins contain consensus sites for N-glycosylation, indicating that these proteins do not traverse the ER-Golgi network. However, there is abundant evidence for the extracellular localisation of some galectins at cell surfaces, in the extracellular matrix and in cell secretions consistent with other evidence for extracellular roles of galectins as modulators of cell adhesion and signalling. How then are galectins secreted if not through the classical secretory pathway? Do all galectins share the same secretory pathway? Can a particular galectin utilise more than one secretory pathway? If galectins play important extracellular roles how is their secretion regulated in relation to function? These are still largely unanswered questions but recent studies are beginning to give glimpses into some novel aspects of the secretion of these intriguing proteins.     

Unconventional protein secretion: an evolving mechanism

The process by which proteins are secreted without entering the classical endoplasmic reticulum (ER)–Golgi complex pathway, in eukaryotic cells, is conveniently called unconventional protein secretion. Recent studies on one such protein called Acb1 have revealed a number of components involved in its secretion. Interestingly, conditions that promote the secretion of Acb1 trigger the biogenesis of a new compartment called CUPS (Compartment for Unconventional Protein Secretion). CUPS form near the ER exit site but lack ER‐specific proteins. Other proteins that share some of the features common with the secretion of Acb1 are interleukin‐1β and tissue transglutaminase. Here I will review recent advances made in the field and propose a new model for unconventional protein secretion.     

Glycosylation and structure of the yeast MF alpha 1 alpha-factor precursor is important for efficient transport through the secretory pathway

The MF alpha 1 gene encodes a precursor, prepro-alpha-factor, that undergoes several proteolytic processing steps within the classical secretory pathway to produce the mature peptide pheromone, alpha-factor. To investigate the role of structural features of the MF alpha 1 precursor in alpha-factor production, we analyzed the effect of mf alpha 1 mutations that alter precursor structure in a number of ways. These mutations resulted in decreased alpha-factor secretion and intracellular accumulation of pro-alpha-factor. With the exception of the mutant lacking all three N glycosylation sites, the pro-alpha-factor forms that accumulated were core glycosylated but had not yet undergone the addition of outer chain carbohydrate. The delay, therefore, occurred at a step prior to the first proteolytic processing step involved in maturation of the precursor and was probably due to inefficient endoplasmic reticulum-to-Golgi transport. Elimination of all three N-glycosylation sites caused a delay in disappearance of intracellular precursor, and alpha-factor secretion was also slowed. These data indicate that N glycosylation is important but not essential for transport of the precursor through the secretory pathway. The decreased alpha-factor secretion and increased precursor accumulation seen with many different structural changes of pro-alpha-factor indicate that the secretory pathway is extremely sensitive to changes in precursor structure. This sensitivity could cause inefficient secretion of heterologous proteins and hybrids between MF alpha 1 and heterologous proteins in yeast cells.     

Identification of human T cell leukemia virus type 1 tax amino acid signals and cellular factors involved in secretion of the viral oncoprotein

Human T cell leukemia virus type 1 (HTLV-1) is the etiologic agent of a number of pathologic abnormalities, including adult T cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The viral oncoprotein Tax has been implicated in the pathogenesis of these diseases. Recently, cell-free Tax was detected in the cerebrospinal fluid of HAM/TSP patients, implying that extracellular Tax may be relevant to neurologic disease. Additionally, the presence of a nuclear export signal within Tax and its active secretion has been demonstrated in vitro. However, the mechanism of Tax secretion remains to be established. Studies reported herein elucidate the process of Tax secretion and identify domains of Tax critical to its subcellular localization and secretion. Tax was shown to interact with a number of cellular secretory pathway proteins in both the model cell line BHK (baby hamster kidney)-21 and an HTLV-1-infected T cell line, C8166, physiologically relevant to HTLV-1-induced disease. Silencing of selected components of the secretory pathway affected Tax secretion, further confirming regulated secretion of Tax. Additionally, mutations in two putative secretory signals within Tax DHE and YTNI resulted in aberrant subcellular localization of Tax and significantly altered protein secretion. Together, these studies demonstrate that Tax secretion is a regulated event facilitated by its interactions with proteins of the cellular secretory pathway and the presence of secretory signals within the carboxyl-terminal domain of the protein.     

Secretory trafficking in neuronal dendrites

The neuronal secretory pathway represents the intracellular route for proteins involved in synaptic transmission and plasticity, as well as lipids required for outgrowth and remodelling of dendrites and axons. Although neurons use the same secretory compartments as other eukaryotic cells, the enormous distances involved, as well as the unique morphology of the neuron and its signalling requirements, challenge canonical models of secretory pathway organization. Here, we review evidence for a distributed secretory pathway in neurons, suggest mechanisms that may regulate secretory compartment distribution, and discuss the implications of a distributed secretory pathway for neuronal morphogenesis and neural-circuit plasticity.     

A new pathway for protein export in Saccharomyces cerevisiae

Several physiologically important proteins lack a classical secretory signal sequence, yet they are secreted from cells. To investigate the secretion mechanism of such proteins, a representative mammalian protein that is exported by a nonclassical mechanism, galectin-1, has been expressed in yeast. Galectin-1 is exported across the yeast plasma membrane, and this export does not require the classical secretory pathway nor the yeast multidrug resistance-like protein Ste6p, the transporter for the peptide a factor. A screen for components of the export machinery has identified genes that are involved in nonclassical export. These findings demonstrate a new pathway for protein export that is distinct from the classical secretory pathway in yeast.