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

A novel membrane structure called CUPS is assembled during the secretion of unconventional cargo such as Acb1

Fus-Mid-GFP and

<p><b>Pma1 localization is defective in </b><b><i>sec14^ts</i></b><b> cells and restored by expression of Sec14^G266D. </b><i>A, sec14^ts</i> cells expressing Fus-Mid-GFP and also containing empty vector, a vector expressing wild type Sec14 on a low copy plasmid, or Sec14^G266D on a high copy (2 μ) plasmid, were grown at 25°C in 1% raffinose containing medium to mid-logarithmic phase. Cells were shifted to 37°C in pre-warmed 2% galactose containing medium for 3 hours. <i>B</i>, cells from <i>A</i> were quantified based on having only plasma membrane (PM) localization, only internal localization or both (vector n = 153, Sec14 n = 73, Sec14^G266D n = 107) <i>C</i>, the wild type <i>SEC14</i> gene was replaced with the <i>sec14^ts</i> allele in a yeast strain expressing chimeric Pma1-RFP. The strain was transformed with either empty vector, a plasmid carried at low copy (ARS/CEN) containing wild type Sec14, and low and high copy (2 μ) plasmids containing Sec14^G266D. Cells were grown at 25°C to mid-logarithmic phase and then transferred to 37°C for 16 hrs subsequent to determination Pma1-RFP localization by fluorescence microscopy.</p

<i>sec14^ts</i> suppressing gene deletions.

<p><i>sec14^ts</i> suppressing gene deletions.</p

The levels of Sec14^G266D is regulated by the proteosome.

<p><i>A</i>, the level of Sec14 and Sec14^G266D in <i>sec14^ts rpn4</i>Δ cells. <i>B</i>, the level of Sec14 and Sec14^G266D in <i>sec14^ts</i> cells treated with MG132. Strains were transformed with plasmids expressing Sec14 or Sec14^G266D containing an N-terminal T7 epitope tag and were grown to mid-logarithmic phase at 25°C, with a subset shifted to 37°C for 2 hours (A). For MG132 treatment cells were grown as before and shifted to 37°C in the presence of 100 μM MG132 for 2 hours. Cells were disrupted by three passes through a French press and unbroken cells removed by centrifugation. Protein extract was separated by SDS-PAGE, transferred to PVDF membrane, and western blots versus the T7 epitope were performed. Pgk1 was used as load control.</p

Known vesicular trafficking pathways are still aberrant in growing cells expressing Sec14^G266D.

<p>The <i>sec14^ts</i> strain transformed with either empty vector, a plasmid carried at low copy (ARS/CEN) containing wild type Sec14, or a high copy (2 μ) plasmid containing Sec14^G266D were grown at 25°C to mid-logarithmic phase and then transferred to 37°C for 1 hr subsequent to determination of: <i>A</i>, invertase secretion (mean ± SE of three separate experiments performed in duplicate), <i>B</i>, or internal retention of Bgl2 at 2 and 16 hrs, similar results were seen at both time point with the 2 hr time point shown. <i>C</i>, the <i>sec14^ts</i> strain containing plasmid borne GFP-Snc1 was transformed with either empty vector, a plasmid carried at low copy (<i>ARS/CEN</i>) containing wild type Sec14, or a high copy (2 μ) plasmid containing Sec14^G266D. Cells were grown at 25°C to mid-logarithmic phase and then transferred to 37°C for 2 hrs. The localization of GFP-Snc1 was determined by fluorescence microscopy in live cells. <i>D</i>, The strains were grown at 25°C to mid-logarithmic phase and then transferred to 37°C for 15 min prior to the addition of FM4-64. The trafficking of FM4-64 in live cells was visualized by fluorescence microscopy.</p

Effect of temperature on Sec14 and Sec14 ^G266D protein levels.

<p><i>A</i>, the <i>sec14^ts</i> cells were transformed with a plasmid expressing Sec14 containing an N-terminal T7 epitope, untagged Sec14, or empty vector. <i>SEC14</i> expression was driven by the constitutive <i>GPD1</i> promoter. Cells were grown in solution at 25°C to mid-logarithmic phase, and serial dilutions of identical numbers of cells were spotted onto plates and incubated at 37°C for two days. <i>B</i>, cells expressing T7-Sec14 or Sec14^G266D were grown to mid-logarithmic phase at 25°C, with a subset shifted to 37°C for 2 hrs. Cells were disrupted by three passes through a French press and membranes were separated from soluble proteins by differential centrifugation. Proteins in each fraction were separated by SDS-PAGE, transferred to PVDF membrane, and western blots were performed. In the blots shown 10 fold more protein extract was loaded in each Sec14^G266D lane compared to extracts containing wild type Sec14 for blots versus the T7 epitope due to protein expression level differences.</p

Membranes accumulate in growing cells expressing Sec14^G266D.

<p>Wild type cells and <i>sec14^ts</i> cells containing empty vector, a vector expressing wild type Sec14 on a low copy plasmid, or Sec14^G266D on a high copy (2 μ) plasmid, were grown at 25°C to mid-logarithmic phase and an aliquot transferred to 37°C for 1 hr followed by incubation in 1.5% KMnO₄, 1% sodium periodate, and then 1% NH₄Cl subsequent to embedding and viewing by transmission electron microscopy.</p

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.We acknowledge support from the Spanish Ministry of Economy and Competitiveness through the program Centro de Excelencia Severo Ochoa 2013–2017 (SEV-2012-0208) and from the Centres de Recerca de Catalunya Program/Generalitat de Catalunya. Vivek Malhotra is an Institució Catalana de Recerca i Estudis Avançats professor at the Center for Genomic Regulation, and the work in his laboratory is funded by grants from the Spanish Ministry of Economy and Competitiveness’s Plan Nacional (BFU2013-44188-P) and Consolider (CSD2009-00016) as well as by the European Research Council (268692). The project has received research funding from the European Union. This paper reflects only the authors’ views. The European Union is not liable for any use that may be made of the information contained therein

Reactive oxygen species triggers unconventional secretion of antioxidants and Acb1

Nutrient deprivation triggers the release of signal-sequence-lacking Acb1 and the antioxidant superoxide dismutase 1 (SOD1). We now report that secreted SOD1 is functionally active and accompanied by export of other antioxidant enzymes such as thioredoxins (Trx1 and Trx2) and peroxiredoxin Ahp1 in a Grh1-dependent manner. Our data reveal that starvation leads to production of nontoxic levels of reactive oxygen species (ROS). Treatment of cells with N-acetylcysteine (NAC), which sequesters ROS, prevents antioxidants and Acb1 secretion. Starved cells lacking Grh1 are metabolically active, but defective in their ability to regrow upon return to growth conditions. Treatment with NAC restored the Grh1-dependent effect of starvation on cell growth. In sum, starvation triggers ROS production and cells respond by secreting antioxidants and the lipogenic signaling protein Acb1. We suggest that starvation-specific unconventional secretion of antioxidants and Acb1-like activities maintain cells in a form necessary for growth upon their eventual return to normal conditions.This work was funded by grants from the Spanish Ministry of Economy and Competitiveness (BFU2013-44188-P and BFU2016_75372-P to V. Malhotra). We acknowledge support of the Spanish Ministry of Economy, Industry and Competitiveness to the European Molecular Biology Laboratories (EMBL) partnership, the Programmes “Centro de Excelencia Severo Ochoa 2013–2017” (SEV-2012-0208 and SEV-2013-0347), and the Centres de Recerca de Catalunya (CERCA) Program/Generalitat de Catalunya. The mass spectrometry data were acquired at the Centre for Genomic Regulation/UPF Proteomics Unit, which is part of Proteored, PRB3, and is funded by El Instituto de Salud Carlos III (ISCIII) and European Regional Development Fund (ERDF; grant PT17/0019 of the PE I+D+i 2013–2016)

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