Oocytes maintain ROS-free mitochondrial metabolism by suppressing complex I.

Oocytes form before birth and remain viable for several decades before fertilization1. Although poor oocyte quality accounts for most female fertility problems, little is known about how oocytes maintain cellular fitness, or why their quality eventually declines with age2. Reactive oxygen species (ROS) produced as by-products of mitochondrial activity are associated with lower rates of fertilization and embryo survival3-5. Yet, how healthy oocytes balance essential mitochondrial activity with the production of ROS is unknown. Here we show that oocytes evade ROS by remodelling the mitochondrial electron transport chain through elimination of complex I. Combining live-cell imaging and proteomics in human and Xenopus oocytes, we find that early oocytes exhibit greatly reduced levels of complex I. This is accompanied by a highly active mitochondrial unfolded protein response, which is indicative of an imbalanced electron transport chain. Biochemical and functional assays confirm that complex I is neither assembled nor active in early oocytes. Thus, we report a physiological cell type without complex I in animals. Our findings also clarify why patients with complex-I-related hereditary mitochondrial diseases do not experience subfertility. Complex I suppression represents an evolutionarily conserved strategy that allows longevity while maintaining biological activity in long-lived oocytes.

Comparative analysis of vertebrates reveals that mouse primordial oocytes do not contain a Balbiani body.

Oocytes spend the majority of their lifetime in a primordial state. The cellular and molecular biology of primordial oocytes is largely unexplored; yet, it is necessary to study them to understand the mechanisms through which oocytes maintain cellular fitness for decades, and why they eventually fail with age. Here, we develop enabling methods for live-imaging-based comparative characterization of Xenopus, mouse and human primordial oocytes. We show that primordial oocytes in all three vertebrate species contain active mitochondria, Golgi and lysosomes. We further demonstrate that human and Xenopus oocytes have a Balbiani body characterized by a dense accumulation of mitochondria in their cytoplasm. However, despite previous reports, we did not find a Balbiani body in mouse oocytes. Instead, we demonstrate that what was previously used as a marker for the Balbiani body in mouse primordial oocytes is in fact a ring-shaped Golgi that is not functionally associated with oocyte dormancy. This study provides the first insights into the organization of the cytoplasm in mammalian primordial oocytes, and clarifies the relative advantages and limitations of choosing different model organisms for studying oocyte dormancy.

Epistemic Standards for Participatory Technology Assessment: Suggestions Based Upon Well-Ordered Science.

When one wants to use citizen input to inform policy, what should the standards of informedness on the part of the citizens be? While there are moral reasons to allow every citizen to participate and have a voice on every issue, regardless of education and involvement, designers of participatory assessments have to make decisions about how to structure deliberations as well as how much background information and deliberation time to provide to participants. After assessing different frameworks for the relationship between science and society, we use Philip Kitcher's framework of Well-Ordered Science to propose an epistemic standard on how citizen deliberations should be structured. We explore what potential standards follow from this epistemic framework focusing on significance versus scientific and engineering expertise. We argue that citizens should be tutored on the historical context of why scientific questions became significant and deemed scientifically and socially valuable, and if citizens report that they are capable of weighing in on an issue then they should be able to do so. We explore what this standard can mean by looking at actual citizen deliberations tied to the 2014 NASA ECAST Asteroid Initiative Citizen forums. We code different vignettes of citizens debating alternative approaches for Mars exploration based upon what level of information seemed to be sufficient for them to feel comfortable in making a policy position. The analysis provides recommendations on how to design and assess future citizen assessments grounded in properly conveying the historical value context surrounding a scientific issue and trusting citizens to seek out sufficient information to deliberate.

Sphingomyelin organization is required for vesicle biogenesis at the Golgi complex.

Sphingomyelin and cholesterol can assemble into domains and segregate from other lipids in the membranes. These domains are reported to function as platforms for protein transport and signalling. Do similar domains exist in the Golgi membranes and are they required for protein secretion? We tested this hypothesis by using D-ceramide-C6 to manipulate lipid homeostasis of the Golgi membranes. Lipidomics of the Golgi membranes isolated from D-ceramide-C6-treated HeLa cells revealed an increase in the levels of C6-sphingomyelin, C6-glucosylceramide, and diacylglycerol. D-ceramide-C6 treatment in HeLa cells inhibited transport carrier formation at the Golgi membranes without affecting the fusion of incoming carriers. The defect in protein secretion as a result of D-ceramide-C6 treatment was alleviated by knockdown of the sphingomyelin synthases 1 and 2. C6-sphingomyelin prevented liquid-ordered domain formation in giant unilamellar vesicles and reduced the lipid order in the Golgi membranes of HeLa cells. These findings highlight the importance of a regulated production and organization of sphingomyelin in the biogenesis of transport carriers at the Golgi membranes.

Early healing assessment with optical coherence tomography of everolimus-eluting stents with bioabsorbable polymer (synergy™) at 3 and 6 months after implantation.

OBJECTIVES: In this study we sought to evaluate coverage and apposition of Synergy™ stent at 3 and 6 months after implantation. BACKGROUND: The Pt-Cr everolimus-eluting stent with abluminal bioabsorbable polymer (Synergy™) is a new generation drug-eluting stent with features potentially favoring an early healing process which could make safe a shorter period of dual antiplatelet-therapy treatment. METHODS: Prospective, two-centers study enrolling patients with similar lesions treated with Synergy™ stents undergoing examination with OCT at 3 and 6 months in the respective centers. Blinded analysis was done at a core lab. Co-primary endpoints were proportion of struts with coverage and with apposition at 3 and 6 months. RESULTS: Finally, 22 patients (30 stents) in the 3 months group and 20 patients (30 stents) in the 6 months group were included. There were no significant differences between groups regarding clinical, angiographic measurements, and procedural data. The rate of strut coverage was 94.5% at 3 months and 96.6% at 6 months (P < 0.001), the rates of apposition were 93.8% and 96.2%, respectively, (P < 0.001), the proportion of uncovered but apposed struts was 2.5% and 1.9% (P = 0.03) and the proportion of uncovered and malapposed struts was 3% and 1.8%, respectively (P < 0.001). The maximal area of malapposition related with uncovered struts was 0.43 ± 0.4 mm(2) at 3 months and 0.14 ± 0.2 mm(2) at 6 months (P = 0.001). CONCLUSIONS: The everolimus-eluting stent with absorbable polymer, Synergy™, is associated to a high degree of intimal coverage and apposition at 3 months after implantation with additional increase at 6 months. © 2015 Wiley Periodicals, Inc.

Accuracy of the angiography-based quantitative flow ratio in intermediate left main coronary artery lesions and comparison with visual estimation.

BACKGROUND: Revascularization of left main coronary artery (LMCA) stenosis is mostly based on angiography. Indices based on angiography might increase accuracy of the decision, although they have been scarcely used in LMCA. The objective of this study is to study the diagnostic agreement of QFR (quantitative flow ratio) with wire-based fractional flow reserve (FFR) in LMCA lesions and to compare with visual severity assessment. METHODS: In a series of patients with invasive FFR assessment of intermediate LMCA stenoses we retrospectively compared the measured value of QFR with that of FFR and the estimate of significance from angiography. RESULTS: 107 QFR studies were included. The QFR intra-observer and inter-observer agreement was 87% and 82% respectively. The mean QFR-FFR difference was 0.047 ± 0.05 with a concordance of 90.7%, sensitivity 88.1%, specificity 92.3%, positive predictive value 88.1% and negative predictive value 92.3%. All these values were superior to those observed with the visual estimation which showed an intra- and inter-observer agreement of 73% and 72% respectively, besides 78% with the FFR value. The low diagnostic performance of the visual estimation and the acceptable performance of the QFR index measurement were observed in all subgroups analysed. CONCLUSIONS: QFR allows an acceptable estimate of the FFR obtained with intracoronary pressure guidewire in intermediate LMCA lesions, and clearly superior to the assessment based on angiography alone. The decision to revascularize patients with moderate LMCA lesions should not be based solely on the degree of angiographic stenosis.

Circulating SOD2 Is a Candidate Response Biomarker for Neoadjuvant Therapy in Breast Cancer.

There is a great need for non-invasive tools that inform of an early molecular response to cancer therapeutic treatment. Here, we tested the hypothesis that proteolytically resistant proteins could be candidate circulating tumor biomarkers for cancer therapy. Proteins resistant to proteolysis are drastically under-sampled by current proteomic workflows. These proteins could be reliable sensors for the response to therapy since they are likely to stay longer in circulation. We selected manganese superoxide dismutase (SOD2), a mitochondrial redox enzyme, from a screening of proteolytic resistant proteins in breast cancer (BC). First, we confirmed the robustness of SOD2 and determined that its proteolytic resistance is mediated by its quaternary protein structure. We also proved that the release of SOD2 upon chemotherapy treatment correlates with cell death in BC cells. Then, after confirming that SOD2 is very stable in human serum, we sought to measure its circulating levels in a cohort of BC patients undergoing neoadjuvant therapy. The results showed that circulating levels of SOD2 increased when patients responded to the treatment according to the tumor shrinkage during neoadjuvant chemotherapy. Therefore, the measurement of SOD2 levels in plasma could improve the non-invasive monitoring of the therapeutic treatment in breast cancer patients. The identification of circulating biomarkers linked to the tumor cell death induced by treatment could be useful for monitoring the action of the large number of cancer drugs currently used in clinics. We envision that our approach could help uncover candidate tumor biomarkers to measure a tumor's response to cancer therapy in real time by sampling the tumor throughout the course of treatment.

The function of GORASPs in Golgi apparatus organization in vivo.

In vitro experiments have shown that GRASP65 (GORASP1) and GRASP55 (GORASP2) proteins function in stacking Golgi cisternae. However, in vivo depletion of GORASPs in metazoans has given equivocal results. We have generated a mouse lacking both GORASPs and find that Golgi cisternae remained stacked. However, the stacks are disconnected laterally from each other, and the cisternal cross-sectional diameters are significantly reduced compared with their normal counterparts. These data support earlier findings on the role of GORASPs in linking stacks, and we suggest that unlinking of stacks likely affects dynamic control of COPI budding and vesicle fusion at the rims. The net result is that cisternal cores remain stacked, but cisternal diameter is reduced by rim consumption.

Regulation of protein transport from the Golgi complex to the endoplasmic reticulum by CDC42 and N-WASP.

Actin is involved in the organization of the Golgi complex and Golgi-to-ER protein transport in mammalian cells. Little, however, is known about the regulation of the Golgi-associated actin cytoskeleton. We provide evidence that Cdc42, a small GTPase that regulates actin dynamics, controls Golgi-to-ER protein transport. We located GFP-Cdc42 in the lateral portions of Golgi cisternae and in COPI-coated and non-coated Golgi-associated transport intermediates. Overexpression of Cdc42 and its activated form Cdc42V12 inhibited the retrograde transport of Shiga toxin from the Golgi complex to the ER, the redistribution of the KDEL receptor, and the ER accumulation of Golgi-resident proteins induced by the active GTP-bound mutant of Sar1 (Sar1[H79G]). Coexpression of wild-type or activated Cdc42 and N-WASP also inhibited Golgi-to-ER transport, but this was not the case in cells expressing Cdc42V12 and N-WASP(Delta WA), a mutant form of N-WASP that lacks Arp2/3 binding. Furthermore, Cdc42V12 recruited GFP-N-WASP to the Golgi complex. We therefore conclude that Cdc42 regulates Golgi-to-ER protein transport in an N-WASP-dependent manner.

Myosin motors and not actin comets are mediators of the actin-based Golgi-to-endoplasmic reticulum protein transport.

We have previously reported that actin filaments are involved in protein transport from the Golgi complex to the endoplasmic reticulum. Herein, we examined whether myosin motors or actin comets mediate this transport. To address this issue we have used, on one hand, a combination of specific inhibitors such as 2,3-butanedione monoxime (BDM) and 1-[5-isoquinoline sulfonyl]-2-methyl piperazine (ML7), which inhibit myosin and the phosphorylation of myosin II by the myosin light chain kinase, respectively; and a mutant of the nonmuscle myosin II regulatory light chain, which cannot be phosphorylated (MRLC2(AA)). On the other hand, actin comet tails were induced by the overexpression of phosphatidylinositol phosphate 5-kinase. Cells treated with BDM/ML7 or those that express the MRLC2(AA) mutant revealed a significant reduction in the brefeldin A (BFA)-induced fusion of Golgi enzymes with the endoplasmic reticulum (ER). This delay was not caused by an alteration in the formation of the BFA-induced tubules from the Golgi complex. In addition, the Shiga toxin fragment B transport from the Golgi complex to the ER was also altered. This impairment in the retrograde protein transport was not due to depletion of intracellular calcium stores or to the activation of Rho kinase. Neither the reassembly of the Golgi complex after BFA removal nor VSV-G transport from ER to the Golgi was altered in cells treated with BDM/ML7 or expressing MRLC2(AA). Finally, transport carriers containing Shiga toxin did not move into the cytosol at the tips of comet tails of polymerizing actin. Collectively, the results indicate that 1) myosin motors move to transport carriers from the Golgi complex to the ER along actin filaments; 2) nonmuscle myosin II mediates in this process; and 3) actin comets are not involved in retrograde transport.

A diacidic motif determines unconventional secretion of wild-type and ALS-linked mutant SOD1.

The nutrient starvation-specific unconventional secretion of Acb1 in Saccharomyces cerevisiae requires ESCRT-I, -II, and -III and Grh1. In this study, we report that another signal sequence lacking cytoplasmic protein, superoxide dismutase 1 (SOD1), and its mutant form linked to amyotrophic lateral sclerosis (ALS), is also secreted by yeast upon nutrient starvation in a Grh1- and ESCRT-I-, -II-, and -III-dependent process. Our analyses reveal that a conserved diacidic motif (Asp-Glu) in these proteins is necessary for their export. Importantly, secretion of wild-type human SOD1 and the ALS-linked mutant in human cells also require the diacidic residues. Altogether, these findings reveal information encoded within the cytoplasmic proteins required for their unconventional secretion and provide a means to unravel the significance of the cytoplasmic versus the secreted form of mutant SOD1 in the pathology of ALS. We also propose how cells, based on a signal-induced change in cytoplasmic physiology, select a small pool of a subset of cytoplasmic proteins for unconventional secretion.

Fluorescent analogues of plasma membrane sphingolipids are sorted to different intracellular compartments in astrocytes; Harmful effects of chronic ethanol exposure on sphingolipid trafficking and metabolism.

Sphingolipids are basic constituents of cellular membranes and are essential for numerous functions such as intracellular signalling. They are transported along the exocytic and endocytic pathways in eukaryotic cells. After endocytosis, fluorescent-labelled sphingolipids are sorted to distinct intracellular organelles prior to recycling (via early/recycling endosomes) or degradation (late endosomes/lysosomes). Here we examine, in primary cultures of rat astrocytes, the internalisation routes followed by C(6)-NBD-glucosylceramide (NBD-GlcCer) and C(6)-NBD-sphingomyelin (NBD-SM) and the effects of ethanol on their endocytic trafficking. Endocytosed plasma membrane NBD-GlcCer and NBD-SM are diverted to the Golgi apparatus and lysosomes, respectively. These different internalisation pathways are maintained regardless of the differentiation stage of astrocytes. Chronic ethanol exposure did not alter this endocytic sorting, but delayed the internalisation of both NBD-sphingolipids. Moreover, ethanol also stimulated the in situ metabolism of NBD-ceramide to NBD-GlcCer and NBD-SM. We conclude that in rat astrocytes internalised plasma membrane NBD-sphingolipids are sorted to different subcellular compartments. The exposure to chronic ethanol perturbed the lipid endocytic process and stimulated the de novo synthesis of NBD-sphingolipids, shifting the balance of sphingolipid metabolism in favour of the sphingomyelin pathway.

Coronary stent implantation in patients older than 75 years of age: clinical profile and initial and long-term (3 years) outcome.

OBJECTIVE: The objective of this study was to compare the initial and long-term outcome of elderly and younger patients after coronary stent implantation. METHODS: The evolutions of 76 patients aged >75 years and of 860 patients aged < or =75 years who underwent consecutive stenting (from June 1991 to June 1997) were compared in a cohort study. RESULTS: The elderly patients had lower left ventricular ejection fractions (0.58 +/- 0.14 vs 0.61 +/- 0.13; P =.03) and more frequently had unstable angina (78.9% vs 55.3%; P <.0001), previous heart failure (10.5% vs 4.9%; P =.03), and multivessel disease (68.4% vs 58.3%; P =.08). After the procedure, the elderly patients showed a higher inhospital mortality rate (6.6% vs 2.4%; P =.03) and myocardial infarction rate (5.3% vs 1.7%; P =.04). The long-term follow-up period (mean, 3.2 +/- 1.4 years; median, 3.0 years) showed in the elderly a higher mortality rate (15.4% vs 5.8%; P =.006), a lower rate of repeat revascularization (9.2% vs 19.7%; P =.04), and a similar incidence rate of major adverse cardiac events (27.7% vs 28.2%; P =.93). Multivariate analysis of the elderly group identified female gender (hazard ratio, 2.19; 95% CI, 1.18 to 4.06; P =.012) and presence of multivessel disease (hazard ratio, 2.35; 95% CI, 1.05 to 5.26; P =.037) as independent predictors of further events. CONCLUSION: Patients aged >75 years have a less favorable baseline profile and higher inhospital and 3-year mortality rates. However, the incidence rate of major adverse cardiac events in the long term is acceptable and similar to that of younger patients.

[Spanish Registry on Cardiac Catheterization Interventions. 11th official report of the Working Group on Cardiac Catheterization and Interventional Cardiology of the Spanish Society of Cardiology (years 1990-2001)]. / Registro Español de Hemodinámica and Cardiología Intervencionista. XI informe oficial de la sección de hemodinámica y cardiología intervendionista de la Sociedad Española de Cardiología (años 1990-2001).

The results of the Spanish Registry of the Working Group on cardiac catheterization and Interventional Cardiology of the Spanish Society of Cardiology (years 1990-2001) are presented. One-hundred-and-three centers contributed data, all the cardiac catheterization laboratories in Spain; 97 centers performed mainly adult catheterization and 6 carried out only pediatric procedures. In 2001, 95,430 diagnostic catheterization procedures were performed, with 79,607 coronary angiograms, representing a total increase of 8.4% over 2000. The population-adjusted incidence was 1947 coronary angiograms per 106 inhabitants. Coronary interventions increased by 15.4% compared with 2000, with a total of 31,290 procedures and an incidence of coronary interventions of 761 per 106 inhabitants. Coronary stents were the most frequently used devices with 39,356 implanted in 2001, and increase of 33.4% over 2000. Stenting accounted for 88.2% of procedures. Direct stenting was done in 11,280 procedures (40.9%). IIb-IIIa glycoprotein inhibitors were given in 7,012 procedures (22.4%). Multivessel percutaneous coronary interventions were performed in 8,445 cases (27%) and interventions were performed ad hoc during diagnostic study in 23,144 cases (74 %).A total of 3,845 percutaneous coronary interventions were carried out in patients with acute myocardial infarction, an increase of 22.9% over 2000 and 12.3% of all interventional procedures. Among non-coronary interventions, atrial septal defect closure was performed more often (161 cases, a 60% increase over 2000). Pediatric interventions increased by 15.4% (from 817 to 943 cases).Lastly, we would like to underline the high rate of reporting by laboratories, which allowed the Registry to compile data that are highly representative of hemodynamic interventions in Spain.

[Spanish Registry on Cardiac Catheterization and Coronary Interventions. Twelfth official report of the Working Group on Cardiac Catheterization and Interventional Cardiology of the Spanish Society of Cardiology (1990-2002)]. / Registro Español de Hemodinámica y Cardiología Intervencionista. XII informe oficial de la Sección de Hemodinámica y Cardiología Intervencionista de la Sociedad Española de Cardiología (1990-2002).

The results of the Registry of the Working Group on Cardiac Catheterization and Interventional Cardiology of the Spanish Society of Cardiology for 2002 are presented. Data were obtained from 101 centers representing all cardiac catheterization laboratories in Spain; 95 centers performed mainly adult catheterization and 6 carried out only pediatric procedures. In 2002, 97,609 diagnostic catheterization procedures were performed, including 83,667 coronary angiograms, representing a total increase of 5.1% in comparison to 2001. The population-adjusted rate was 2,053 coronary angiograms per 106 inhabitants. Coronary interventions increased by 11% in comparison to 2001, with a total of 34,723 procedures and a rate of coronary interventions of 850 per 106 inhabitants. Coronary stents were the devices used most frequently, with 47,249 implanted in 2002, for a total increase of 20% in comparison to 2001. Stenting accounted for 91.7% of all procedures. Direct stenting was done in 13 768 procedures (43.2%). IIb-IIIa glycoprotein inhibitors were used in 9966 procedures (28.7%). Multivessel percutaneous coronary interventions were performed in 9,830 patients (28%), and ad hoc interventions were done in the course of diagnostic coronary angiography in 26,341 patients (76%).A total of 4,766 percutaneous coronary interventions were done in patients with acute myocardial infarction, representing an increase of 23.9% in comparison to 2001, and accounting for 13.7% of all interventional procedures. Of the noncoronary interventions recorded, we note the decrease in percutaneous mitral valvuloplasties (21.2%) and atrial septal defect closures (11.1%), and the slight increase in pediatric interventions (3.7%). In conclusion, we emphasize the high rate of reporting by laboratories, which allows the Registry to compile data that are highly representative of the activity at cardiac catheterization laboratories in Spain

[Coronary angioplasty in diabetic patients. Current and future perspectives]. / Angioplastia coronaria en el paciente diabético. Situación actual y perspectivas futuras.

It has been estimated that 15-25% of patients who undergo percutaneous or surgical coronary angioplasty are diabetics. The indications for coronary revascularization and initial results of the procedure do not differ substantially between patients with diabetes mellitus and non-diabetics. However, the long-term results of both percutaneous and surgical coronary angioplasty are less favorable in diabetics in terms of mortality and the need for new revascularization procedures. The development and widespread use of stents and glycoprotein IIb/IIIa receptor inhibiting drugs have improved the clinical evolution of diabetics treated with angioplasty. Currently available data show that the administration of glycoprotein IIb/IIIa inhibitors to patients undergoing coronary angioplasty is especially useful in diabetics and improves short-term and long-term results, decreasing one-year mortality by 45%. There seem to be indications for the routine use of glycoprotein IIb/IIIa inhibitors in diabetics treated with angioplasty. While the use of stents has improved long-term and short-term results in diabetics, the success rates of angioplasty in diabetics are still lower than in non-diabetics. Diabetes is still an independent predictor of restenosis and long-term events after stenting interventions. Analysis of the studies comparing percutaneous and surgical revascularization in diabetic patients with multivessel disease shows that surgery is superior in terms of long-term mortality and need for new revascularization procedures. Stenting has improved, but not substantially, the results of multivessel angioplasty in diabetics. Therefore, the indications for angioplasty in multivessel diabetics should be evaluated individually. Factors that contribute to the less favorable post-angioplasty evolution of diabetic patients are more rapid progression of atherosclerosis and, especially, a higher rate of restenosis. New angioplasty techniques, such as brachytherapy and drug-eluting stents, are likely to significantly improve the results of percutaneous interventions in diabetics, thus allowing the indications for angioplasty in diabetics to be extended even further in the near future.

[Intracoronary stem cell transplantation in acute myocardial infarction]. / Regeneración micárdica mediante la implantación intracoronaria de células madre en el infarto agudo de miocardio.

INTRODUCTION AND OBJECTIVES: Experimental and clinical studies suggest that necrotic myocardium may have the capacity to regenerate. We have started a clinical study to demonstrate that the intracoronary implantation of stem cells is feasible and safe. The results in our first 5 patients are presented here. PATIENTS AND METHOD: We included patients with anterior acute myocardial infarction and isolated stenosis of the left anterior descending artery that was successfully repaired by primary or facilitated angioplasty. Patients received an intracoronary infusion of bone marrow-derived cells 10-15 days after the infarction. The follow-up protocol included low-dose dobutamine echocardiography, magnetic resonance studies and ECG Holter monitoring. RESULTS: The procedure was carried out with no complications. No patient had a cardiac event during the first 6 months. One patient had a transient ischemic attack without sequelae. No arrhythmias were found. Left ventricular end-diastolic volume remained the same at 6 months (159+/-25 ml, 157+/-16 ml), left ventricular end-systolic volume decreased (77+/-22 ml, 65+/-16 ml), and the ejection fraction increased (53+/-7%, 58+/-8%) although no statistically significant differences were found. In the 3 patients in whom dobutamine echocardiography ruled out viability, we found a significant reduction in both volumes. CONCLUSIONS: Intracoronary bone marrow-derived cell transplantation after an acute myocardial infarction seems to be safe and feasible, and might lead to favorable remodeling.

Remodeling of secretory compartments creates CUPS during nutrient starvation.

Upon starvation, Grh1, a peripheral membrane protein located at endoplasmic reticulum (ER) exit sites and early Golgi in Saccharomyces cerevisiae under growth conditions, relocates to a compartment called compartment for unconventional protein secretion (CUPS). Here we report that CUPS lack Golgi enzymes, but contain the coat protein complex II (COPII) vesicle tethering protein Uso1 and the Golgi t-SNARE Sed5. Interestingly, CUPS biogenesis is independent of COPII- and COPI-mediated membrane transport. Pik1- and Sec7-mediated membrane export from the late Golgi is required for complete assembly of CUPS, and Vps34 is needed for their maintenance. CUPS formation is triggered by glucose, but not nitrogen starvation. Moreover, upon return to growth conditions, CUPS are absorbed into the ER, and not the vacuole. Altogether our findings indicate that CUPS are not specialized autophagosomes as suggested previously. We suggest that starvation triggers relocation of secretory and endosomal membranes, but not their enzymes, to generate CUPS to sort and secrete proteins that do not enter, or are not processed by enzymes of the ER-Golgi pathway of secretion.

Biogenesis of a novel compartment for autophagosome-mediated unconventional protein secretion.

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.