Autophagosome Formation Depends on the Small GTPase Rab1 and Functional ER Exit Sites

Autophagy is an important cellular degradation pathway present in all eukaryotic cells. Via this pathway, portions of the cytoplasm and/or organelles are sequestered in double‐membrane structures called autophagosomes. In spite of the significant advance achieved in autophagy, the long‐standing question about the source of the autophagic membrane remains unsolved. We have investigated the role of the secretory pathway in autophagosome biogenesis. Sar1 and Rab1b are monomeric GTPases that control traffic from the endoplasmic reticulum (ER) to the Golgi. We present evidence indicating that the activity of both proteins is required for autophagosome formation. Overexpression of dominant‐negative mutants and the use of siRNAs impaired autophagosome generation as determined by LC3 puncta formation and light chain 3 (LC3)‐II processing. In addition, our results indicate that the autophagic and secretory pathways intersect at a level preceding the brefeldin A blockage, suggesting that the transport from the cis/medial Golgi is not necessary for autophagosome biogenesis. Our present results highlight the role of transport from the ER in the initial events of the autophagic vacuole development.     

Specific autophagy and ESCRT components participate in the unconventional secretion of CFTR

The most common mutation in cystic fibrosis patients is a phenylalanine deletion at position 508 (ΔF508) in the CFTR (cystic fibrosis transmembrane conductance regulator) gene. This mutation impairs cell-surface trafficking of CFTR. During cellular stress, core-glycosylated CFTRΔF508 is transported to the cell surface from the endoplasmic reticulum (ER) via an unconventional route that bypasses the Golgi. However, the mechanisms for this unconventional secretory pathway of CFTR are not well delineated. Here, we report that components of the macroautophagy/autophagy and ESCRT (endosomal sorting complex required for transport) pathways are involved in unconventional secretion of CFTR. In mammalian cells, we found that autophagic pathways were modulated by conditions that also stimulate unconventional secretion, namely ER stress and an ER-to-Golgi transport blockade. Additionally, we found that knockdown of early autophagy components, ATG5 and ATG7, and treatment with pharmacological autophagy inhibitors, wortmannin and 3-methyladenine, abolished the unconventional secretion of CFTR that had been stimulated by ER stress and an ER-to-Golgi blockade. Interestingly, immunoelectron microscopy revealed that GORASP2/GRASP55, which mediates unconventional CFTR trafficking, is present in multivesicular bodies (MVB) and autophagosomal structures under ER stress conditions. A custom small-interfering RNA screen of mammalian ESCRT proteins that mediate MVB biogenesis showed that silencing of some ESCRTs, including MVB12B, inhibited unconventional CFTRΔF508 secretion. Furthermore, MVB12B overexpression partially rescued cell-surface expression and Cl^? channel function of CFTRΔF508. Taken together, these results suggest that components involved in early autophagosome formation and the ESCRT/MVB pathway play a key role in the stress-induced unconventional secretion of CFTR.     

Non-classical membrane trafficking processes galore

Dogmatic views of how proteins and other cellular components may traffic within and between eukaryotic cells have been challenged in the past few years. Beyond the classical secretory/exocytic pathway and its established players, other pathways of cell surface membrane transport, generally termed “unconventional secretion,” are now better understood. More insights have also been gleaned on the roles of secreted or shedding microvesicles, either exosomal or ectosomal in origin, in unconventional secretion. Recent works have also revealed key molecular components, particularly the Golgi reassembly stacking protein (GRASP), and the importance of stress‐induced autophagy, in unconventional exocytic transport. This GRASP and autophagy‐dependent (GAD) mode appears to underlie the unconventional exocytosis of many soluble and membrane cargoes. Likewise, recent findings have revealed transport processes that contrast the classically known mitochondria import, namely vesicular transport from the mitochondria to peroxisomes and lysosomes. Mitochondria‐peroxisomal targeting of mitochondria‐derived vesicles appears to involve the retromer complex, which was classically associated with endosome‐Golgi membrane traffic. The routes of intracellular membrane transport and communications between eukaryotic organelles now appear far more complex that one would have imagined 10 years ago. J. Cell. Physiol. 227: 3722–3730, 2012. © 2012 Wiley Periodicals, Inc.     

A positive signal prevents secretory membrane cargo from recycling between the Golgi and the ER

The Golgi complex and ER are dynamically connected by anterograde and retrograde trafficking pathways. To what extent and by what mechanism outward‐bound cargo proteins escape retrograde trafficking has been poorly investigated. Here, we analysed the behaviour of several membrane proteins at the ER/Golgi interface in live cells. When Golgi‐to‐plasma membrane transport was blocked, vesicular stomatitis virus glycoprotein (VSVG), which bears an ER export signal, accumulated in the Golgi, whereas an export signal‐deleted version of VSVG attained a steady state determined by the balance of retrograde and anterograde traffic. A similar behaviour was displayed by EGF receptor and by a model tail‐anchored protein, whose retrograde traffic was slowed by addition of VSVG's export signal. Retrograde trafficking was energy‐ and Rab6‐dependent, and Rab6 inhibition accelerated signal‐deleted VSVG's transport to the cell surface. Our results extend the dynamic bi‐directional relationship between the Golgi and ER to include surface‐directed proteins, uncover an unanticipated role for export signals at the Golgi complex, and identify recycling as a novel factor that regulates cargo transport out of the early secretory pathway.     

Distinct retrieval and retention mechanisms are required for the quality control of endoplasmic reticulum protein folding.

Proteins destined for the secretory pathway must first fold and assemble in the lumen of endoplasmic reticulum (ER). The pathway maintains a quality control mechanism to assure that aberrantly processed proteins are not delivered to their sites of function. As part of this mechanism, misfolded proteins are returned to the cytosol via the ER protein translocation pore where they are ubiquitinated and degraded by the 26S proteasome. Previously, little was known regarding the recognition and targeting of proteins before degradation. By tracking the fate of several mutant proteins subject to quality control, we demonstrate the existence of two distinct sorting mechanisms. In the ER, substrates are either sorted for retention in the ER or are transported to the Golgi apparatus via COPII-coated vesicles. Proteins transported to the Golgi are retrieved to the ER via the retrograde transport system. Ultimately, both retained and retrieved proteins converge at a common machinery at the ER for degradation. Furthermore, we report the identification of a gene playing a novel role specific to the retrieval pathway. The gene, BST1, is required for the transport of misfolded proteins to the Golgi, although dispensable for the transport of many normal cargo proteins.     

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.     

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.     

Hidden Behind Autophagy: The Unconventional Roles of ATG Proteins

Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved intracellular catabolic transport route that generally allows the lysosomal degradation of cytoplasmic components, including bulk cytosol, protein aggregates, damaged or superfluous organelles and invading microbes. Target structures are sequestered by double‐membrane vesicles called autophagosomes, which are formed through the concerted action of the autophagy (ATG)‐related proteins. Until recently it was assumed that ATG proteins were exclusively involved in autophagy. A growing number of studies, however, have attributed functions to some of them that are distinct from their classical role in autophagosome biogenesis. Autophagy‐independent roles of the ATG proteins include the maintenance of cellular homeostasis and resistance to pathogens. For example, they assist and enhance the turnover of dead cells and microbes upon their phagocytic engulfment, and inhibit murine norovirus replication. Moreover, bone resorption by osteoclasts, innate immune regulation triggered by cytoplasmic DNA and the ER‐associated degradation regulation all have in common the requirement of a subset of ATG proteins. Microorganisms such as coronaviruses, Chlamydia trachomatis or Brucella abortus have even evolved ways to manipulate autophagy‐independent functions of ATG proteins in order to ensure the completion of their intracellular life cycle. Taken together these novel mechanisms add to the repertoire of functions and extend the number of cellular processes involving the ATG proteins.     

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.     

A Secretory Golgi Bypass Route to the Apical Surface Domain of Epithelial MDCK Cells

Proteins leave the endoplasmic reticulum (ER) for the plasma membrane via the classical secretory pathway, but routes bypassing the Golgi apparatus have also been observed. Apical and basolateral protein secretion in epithelial Madin-Darby canine kidney (MDCK) cells display differential sensitivity to Brefeldin A (BFA), where low concentrations retard apical transport, while basolateral transport still proceeds through intact Golgi cisternae . We now describe that BFA-mediated retardation of glycoprotein and proteoglycan transport through the Golgi apparatus induces surface transport of molecules lacking Golgi modifications, possessing those acquired in the ER. Low concentrations of BFA induces apical Golgi bypass, while higher concentrations were required to induce basolateral Golgi bypass. Addition of the KDEL ER-retrieval sequence to model protein cores allowed observation of apical Golgi bypass in untreated MDCK cells. Basolateral Golgi bypass was only observed after the addition of BFA or upon cholesterol depletion. Thus, in MDCK cells, an apical Golgi bypass route can transport cargo from pre-Golgi organelles in untreated cells, while the basolateral bypass route is inducible.     

Monomerization and ER Relocalization of GRASP Is a Requisite for Unconventional Secretion of CFTR

Induction of endoplasmic reticulum (ER)‐to‐Golgi blockade or ER stress induces Golgi reassembly stacking protein (GRASP)‐mediated, Golgi‐independent unconventional cell‐surface trafficking of the folding‐deficient ΔF508‐cystic fibrosis transmembrane conductance regulator (CFTR). However, molecular mechanisms underlying this process remain elusive. Here, we show that phosphorylation‐dependent dissociation of GRASP homotypic complexes and subsequent relocalization of GRASP to the ER play a critical role in the unconventional secretion of CFTR. Immunolocalization analyses of mammalian cells revealed that the Golgi protein GRASP55 was redistributed to the ER by stimuli that induce unconventional secretion of ΔF508‐CFTR, such as induction of ER‐to‐Golgi blockade by the Arf1 mutant. Notably, the same stimuli also induced phosphorylation of regions near the C‐terminus of GRASP55 and dissociation of GRASP homomultimer complexes. Furthermore, phosphorylation‐mimicking mutations of GRASP55 induced the monomerization and ER relocalization of GRASP55, and these changes were nullified by phosphorylation‐inhibiting mutations. These results provide mechanistic insights into how GRASP accesses the ER‐retained ΔF508‐CFTR and mediates the ER stress‐induced unconventional secretion pathway.      

The role of microtubules in transport between the endoplasmic reticulum and Golgi apparatus in mammalian cells

The organization of intracellular compartments and the transfer of components between them are central to the correct functioning of mammalian cells. Proteins and lipids are transferred between compartments by the formation, movement and subsequent specific fusion of transport intermediates. These vesicles and membrane clusters must be coupled to the cytoskeleton and to motor proteins that drive motility. Anterograde ER (endoplasmic reticulum)-to-Golgi transport, and the converse step of retrograde traffic from the Golgi to the ER, are now known to involve coupling of membranes to the microtubule cytoskeleton. Here we shall discuss our current understanding of the mechanisms that link membrane traffic in the early secretory pathway to the microtubule cytoskeleton in mammalian cells. Recent data have also provided molecular detail of functional co-ordination of motor proteins to specify directionality, as well as mechanisms for regulating motor activity by protein phosphorylation.     

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.     

Exit of GPI‐Anchored Proteins from the ER Differs in Yeast and Mammalian Cells

Previous studies have shown that yeast glycosylphosphatidylinositol‐anchored proteins (GPI‐APs) and other secretory proteins are preferentially incorporated into distinct coat protein II (COPII) vesicle populations for their transport from the endoplasmic reticulum (ER) to the Golgi apparatus, and that incorporation of yeast GPI‐APs into COPII vesicles requires specific lipid interactions. We compared the ER exit mechanism and segregation of GPI‐APs from other secretory proteins in mammalian and yeast cells. We find that, unlike yeast, ER‐to‐Golgi transport of GPI‐APs in mammalian cells does not depend on sphingolipid synthesis. Whereas ER exit of GPI‐APs is tightly dependent on Sar1 in mammalian cells, it is much less so in yeast. Furthermore, in mammalian cells, GPI‐APs and other secretory proteins are not segregated upon COPII vesicle formation, in contrast to the remarkable segregation seen in yeast. These findings suggest that GPI‐APs use different mechanisms to concentrate in COPII vesicles in the two organisms, and the difference might explain their propensity to segregate from other secretory proteins upon ER exit.     

Golgi apparatus-localized synaptotagmin 2 is required for unconventional secretion in Arabidopsis

Background

Most secretory proteins contain signal peptides that direct their sorting to the ER and secreted via the conventional ER/Golgi transport pathway, while some signal-peptide-lacking proteins have been shown to export through ER/Golgi independent secretory pathways. Hygromycin B is an aminoglycoside antibiotic produced by Streptomyces hygroscopicus that is active against both prokaryotic and eukaryotic cells. The hygromycin phosphotransferase (HYG^R) can phosphorylate and inactivate the hygromycin B, and has been widely used as a positive selective marker in the construction of transgenic plants. However, the localization and trafficking of HYG^R in plant cells remain unknown. Synaptotagmins (SYTs) are involved in controlling vesicle endocytosis and exocytosis as calcium sensors in animal cells, while their functions in plant cells are largely unclear.

Methodology/Principal Findings

We found Arabidopsis synaptotagmin SYT2 was localized on the Golgi apparatus by immunofluorescence and immunogold labeling. Surprisingly, co-expression of SYT2 and HYG^R caused hypersensitivity of the transgenic Arabidopsis plants to hygromycin B. HYG^R, which lacks a signal sequence, was present in the cytoplasm as well as in the extracellular space in HYG^R-GFP transgenic Arabidopsis plants and its secretion is not sensitive to brefeldin A treatment, suggesting it is not secreted via the conventional secretory pathway. Furthermore, we found that HYG^R-GFP was truncated at carboxyl terminus of HYG^R shortly after its synthesis, and the cells deficient SYT2 failed to efficiently truncate HYG^R-GFP,resulting in HYG^R-GFP accumulated in prevacuoles/vacuoles, indicating that SYT2 was involved in HYG^R-GFP trafficking and secretion.

Conclusion/Significance

These findings reveal for the first time that SYT2 is localized on the Golgi apparatus and regulates HYG^R-GFP secretion via the unconventional protein transport from the cytosol to the extracelluar matrix in plant cells.     

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.     

真核细胞非经典蛋白分泌途径

在生物体中, 细胞间的信息传递是细胞生长、分化、发育、增殖、凋亡等生命活动的基本保证, 而蛋白分泌是细胞间信息传递的重要方式。大多数分泌蛋白都是通过内质网-高尔基体(ER-Golgi)途径分泌的。然而越来越多的研究表明, 存在着一类无信号肽的分泌蛋白, 这类蛋白不依赖ER-Golgi途径就能分泌到细胞外发挥功能, 被称为非经典分泌蛋白。非经典蛋白的分泌有其特有的机制, 它对ER-Golgi分泌途径是一种必要和有益的补充。非经典分泌与细胞增殖、免疫反应、肿瘤形成、传染病病理学等密切相关。文章旨在对非经典分泌蛋白的特点、分泌机制及生物学意义进行概述。     

Pathways followed by ricin and Shiga toxin into cells

The plant toxin ricin and the bacterial toxin Shiga toxin belong to a group of protein toxins that inhibit protein synthesis in cells enzymatically after entry into the cytosol. Ricin and Shiga toxin, which both have an enzymatically active moiety that inactivates ribosomes and a moiety that binds to cell surface receptors, enter the cytosol after binding to the cell surface, endocytosis by different mechanisms, and retrograde transport to the Golgi apparatus and the endoplasmic reticulum (ER). The toxins can be used to investigate the various transport steps involved, both the endocytic mechanisms as well as pathways for retrograde transport to the ER. Recent studies show that not only do several endocytic mechanisms exist in the same cell, but they are not equally sensitive to removal of cholesterol. New data have revealed that there is also more than one pathway leading from endosomes to the Golgi apparatus and retrogradely from the Golgi to the ER. Trafficking of protein toxins along these pathways will be discussed in the present article.