Ypt31/32 as a secretory vesicle organizer: Interactions with Sec4 and Myo2.

Ypt31/32 as a secretory vesicle organizer: Interactions with Sec4 and Myo2

GFP-Snc1-PEM accumulates in APs of <i>vps21∆</i> mutant cells and macro-ER-phagy is independent of UPR induction.

<p><b>A.</b> The <i>ypt1-1</i> mutation is epistatic to <i>vps21∆</i> in ER-phagy. yDsRed-Snc1-PEM was overexpressed in WT, <i>vps21∆</i>, <i>ypt1-1</i> and <i>ypt1-1 vps21∆</i> double mutant cells that also expressed the autophagosomal marker yEGFP-Atg8. Cells were analyzed by live-cell microscopy. Shown from left to right: DIC, DsRed, GFP, merge, % cells Atg8 dots, number of Atg8 dots per cell, and % cells in which the Atg8 dots co-localize with Snc1-PEM. About 50% of WT and 85% of <i>vps21∆</i> mutant cells contain ~1 dot of Atg8 representing the AP. Importantly, in ~70% of the <i>vps21∆</i> mutant cells Snc1-PEM co-localizes with the APs, as compared to ~4% in WT cells. In contrast, ~90% <i>ypt1-1</i> and <i>ypt1-1 vps21∆</i> mutant cells contain three APs per cell, and Snc1-PEM does not co-localize with them. Arrows point to co-localization; arrowheads point to either Atg8 dots or GFP-Snc1-PEM that do not co-localize. <b>B-D.</b> UPR induction is not required for macro-ER-phagy. The UPR regulators Ire1 or Hac1 were deleted in the WT and <i>ypt1-1</i> mutant cells. The following effects of overexpression of GFP-Snc1-PEM in WT and <i>ypt1-1</i> mutant cells, without and with <i>ire1∆</i> or <i>hac1∆</i>, were analyzed as described for <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005390#pgen.1005390.g001" target="_blank">Fig 1A–1C</a>, respectively: protein level (<b>B</b>), accumulation of GFP-Snc1-PEM in aberrant structures (<b>C</b>), and UPR induction (<b>D</b>). <b>B.</b> Deletion of either Ire1 or Hac1 does not affect the level of Snc1-PEM accumulation in WT or <i>ypt1-1</i> mutant cells. <b>C.</b> Deletion of either Ire1 or Hac1 does not affect the percent of WT and <i>ypt1-1</i> mutant cells that accumulate aberrant intra-cellular Snc1-PEM. Shown from top to bottom: DIC, GFP, and % cells with intracellular Snc1-PEM structures. <b>D.</b> Deletion of either Ire1 or Hac1 obliterate UPR in both WT and <i>ypt1-1</i> mutant cells. +/- and error bars represent STDEV. Results in this figure represent at least two independent experiments.</p

Immunoblot analysis of ER-resident proteins with and without overexpression of GFP-Snc1-PEM.

<p><b>A.</b> A diagram showing two groups of ER-resident proteins: those that become macro-ER-phagy cargos (in green), and those that do not (in blue or grey). Like overexpressed Snc1-PEM or Snq2, the ER-resident membrane proteins Sec61 (translocon subunit) and Hmg1 (sterol biogenesis) are transported to the vacuole via macro-ER-phagy. In contrast, the ER-to-Golgi exit regulators Sec12 and Sec13 and the ER lumen chaperone Kar2 are not co-transported to the lysosome through macro-ER-phagy. Four or six different strains were used for this analysis: WT (<i>YPT1</i>), <i>pep4∆</i>, and <i>pep4 prb1∆</i>, <i>ypt1-1</i>, <i>ypt1-1 pep4∆</i>, and <i>ypt1-1 pep4∆ prb1∆</i>. In each strain, one ER-resident protein was tagged at its C-terminus (except for Kar2). (The <i>pep4∆</i> strains were not used in all experiments because they do not show the full defect). The level of ER-resident proteins was determined by immunoblot analysis in cells that either do not (<b>B-D</b>) or do overexpress GFP-Snc1-PEM (<b>F-H</b>). <b>B-C.</b> Sec61-3xHA (B) and Sec13-3xHA (C) expressing cells (2 independent un-transformed colonies) were tested by immunoblot analysis (using anti-HA antibodies). Shown from top to bottom: strain genotype, HA-tagged protein, G6PDH (loading control), quantification of HA-tagged protein expressed as average fold of WT (p-value = 0.025 for Sec61). <b>D.</b> Summary of immunoblot analyses of ER-resident proteins level in cells that do not overexpress GFP-Snc1-PEM: WT (<i>PEP4 PRB1</i>) vs <i>pep4∆ prb1∆</i> (left), <i>ypt1-1 PEP4 PRB1</i> vs <i>ypt1-1 pep4∆ prb1∆</i> (right). Immunoblots and quantification are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005390#pgen.1005390.g006" target="_blank">Fig 6B–6C</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005390#pgen.1005390.s006" target="_blank">S6A–S6C Fig</a>. In <i>YPT1</i> cells, the levels of Sec61 and Hmg1 (green) are slightly increased (14 and 38%, respectively) in strains defective in vacuolar proteolysis; the levels of Sec12, Sec13 and Kar2 are not increased (blue and grey). The levels of Sec61 and Hmg1 (green) are increased by 2-2.5 fold in <i>ypt1-1</i> mutant cells when compared to WT cells, regardless if they are defective in vacuolar proteolysis or not. In contrast, the levels of Sec12, Sec13 and Kar2 are only slightly increased (blue and grey). <b>E.</b> The level of the ER-resident protein Sec61 is similar whether GFP-Snc1-PEM is overexpressed or not. Wild-type cells were transformed with a 2μ plasmid, either empty or for overexpression of GFP-Snc1-GEM (2 independent transformants). Shown from top to bottom: plasmid, Sec61, G6PDH (loading control), GFP-Snc1-PEM, and quantification of Sec61 expressed as average fold of WT with empty plasmid. <b>F-G.</b> Sec61-3xHA (F) and Sec13-3xHA (G) expressing cells were transformed with a 2μ plasmid for overexpression of GFP-Snc1-PEM. Cell lysates were tested by immunoblot analysis (using ant-HA and anti-GFP antibodies). Shown from top to bottom: strain genotype, the specific ER-resident protein tested, quantification of the ER-resident protein bands expressed as average fold of WT, GFP-Snc1-PEM, quantification of the GFP-Snc1-PEM bands expressed as average fold of WT, and G6PDH (loading control). <b>H.</b> Summary of immunoblot analysis of ER-resident proteins level in cells overexpressing GFP-Snc1-PEM: WT (<i>PEP4 PRB1</i>) vs <i>pep4∆ prb1∆</i> (left), <i>ypt1-1 PEP4 PRB1</i> vs <i>ypt1-1 pep4∆ prb1∆</i> (right). Immunoblots and quantification are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005390#pgen.1005390.g006" target="_blank">Fig 6F and 6G</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005390#pgen.1005390.s006" target="_blank">S6F–S6H Fig</a>. In <i>YPT1</i> cells, like Snc1-PEM (black), the levels of Hmg1 and Sec61 (green) are increased >10 fold in strains defective in vacuolar proteolysis. In contrast, the levels of Sec12, Sec13 and Kar2 are not changed (blue and grey). Like Snc1-PEM (black), the protein levels of Hmg1 and Sec61 (green) are increased ~15 fold in <i>ypt1-1</i> mutant cells when compared to WT cells, regardless if they are defective in vacuolar proteolysis or not. In contrast, the levels of Sec12, Sec13 and Kar2 are only slightly increased (blue and grey). +/- and error bars represent STDEV. Results in this figure represent at least two independent experiments.</p

The general Atgs Atg1 and Atg8 and the selective Atg Atg11, but not other selective Atgs, are required for macro-ER-phagy.

<p><b>A.</b> GFP-Snc1-PEM protein accumulates in <i>atg1∆</i>, <i>atg8∆</i>, and <i>atg11∆</i> mutant cells. Lysates were prepared from wild type (WT), <i>ypt1-1</i> (for comparison), <i>atg1∆</i>, <i>atg8∆</i>, and <i>atg11∆</i> mutant cells transformed with a 2μ plasmid expressing GFP-Snc1-PEM from the TPI promoter. The level of GFP-Snc1-PEM was determined using immunoblot analysis with anti-GFP antibodies. The bands were quantified and increase in the protein level in mutant versus the WT cells is shown under the blot and adjusted to the G6PDH loading control. <b>B.</b> GFP-Snc1-PEM protein accumulates in aberrant ER structures in <i>atg1∆</i>, <i>atg8∆</i>, and <i>atg11∆</i> mutant cells. The ER-marker Sec61 was tagged with mCherry in strains from panel A, and the cells were examined by live-cell microscopy. Shown from left to right: DIC, GFP, mCherry, merge, % cells with intracellular GFP-Snc1-PEM (number of cells with internal GFP-Snc1-PEM / number of cells visualized), and % cells in which intra-cellular Snc1-PEM co-localizes with Sec61. <b>C.</b> UPR is induced in <i>atg1∆</i>, <i>atg8∆</i>, and <i>atg11∆</i> mutant cells. Cells from panel A were transformed with a second plasmid that expresses β-gal from a UPR promoter. UPR was determined and expressed as % of the WT response. <b>D-E.</b> Unlike deletion of Atg11, deletion of other known selective Atgs required for the CVT pathway (Atg19), mitophagy (Atg32) and pexophagy (Atg36), does not result in increase of GFP-Snc1-PEM protein level (<b>D</b>), intra-cellular accumulation of GFP-Snc1-PEM, (<b>E</b>), and induction of the UPR response (<b>F</b>). Wild type (WT), <i>atg19∆</i>, <i>atg11∆</i>, <i>atg32∆</i>, and <i>atg36∆</i> mutant cells overexpressing GFP-Snc1-PEM were analyzed as described for panels A-C, respectively. <b>E.</b> Shown from left to right: DIC, GFP, and % cells with intracellular Snc1-PEM structures. +/- and error bars represent STDEV. Results in this figure represent at least two independent experiments.</p

Live-cell microscopy analysis of ER-resident proteins upon overexpression of GFP-Snc1-PEM.

<p><b>A.</b> In cells defective in vacuolar proteolysis (<i>pep4∆ prb1∆</i>), Hmg1 co-localizes with 70% of intra-cellular Snc1-PEM, but Sec13 does not (at least 36 cells were analyzed for each strain). No co-localization was observed for resident ER proteins and overexpressed Snc1-PEM in WT cells (<i>PEP4 PRB1</i>). Hmg1 (left) and Sec13 (right) were tagged with mCherry at their C-terminus in <i>PEP4 PRB1</i> (WT) and <i>pepb4∆ prb1∆</i> mutant cells. Cells were transformed with a 2μ plasmid for overexpression of GFP-Snc1-PEM and analyzed by live-cell microscopy. Shown from top to bottom: DIC, GFP, mCherry, merge (yellow), % cells with intra-cellular Snc1-PEM, and % co-localization of the ER protein with intra-cellular Snc1-PEM. <b>B.</b> The ER-resident protein Hmg1 is delivered to the vacuole with GFP-Snc1-PEM. The vacuole of <i>PEP4 PRB1</i> (WT) and <i>pepb4∆ prb1∆</i> mutant cells expressing Hmg1-mCherry and overexpressing GFP-Snc1-PEM (see Panel A, left) was stained with CMAC-Arg (blue), and the cells were analyzed by live-cell microscopy. In WT cells (top), there is no intracellular Snc1-PEM and the red-labeled ER is distinguished from the blue vacuole. In contrast, in ~75% <i>pep4∆ prb1∆</i> mutant cells (bottom), which are defective in vacuolar proteolysis, Hmg1 (red) co-localizes with Snc1-PEM (green) in the vacuole (blue). Shown from left to right: DIC, GFP, mCherry, blue, merge (white shows merge of the three colors), % cells with intracellular Snc1-PEM, % cells in which Hmg1 with Snc1-PEM co-localize, and % co-localization of the two proteins with the vacuolar dye (38 cells were analyzed for each strain). <b>C.</b> >80% of <i>ypt1-1</i> mutant cells, regardless if they are <i>PEP4 PRB1</i> or <i>pep4∆ prb1∆</i>, accumulate aberrant GFP-Snc1-PEM structures. Hmg1 co-localizes with >70% of these structures, whereas Sec13 co-localizes with only ~15% of these structures (36–43 cells were analyzed for each strain). The experiment was done as described for Panel A. <b>D.</b> Summary of microscopy analysis of ER-resident proteins co-localization with GFP-Snc1-PEM in cells overexpressing GFP-Snc1-PEM: WT (<i>PEP4 PRB1</i>) vs <i>pep4∆ prb1∆</i> (left), and <i>ypt1-1 PEP4 PRB1</i> vs <i>ypt1-1 pep4∆ prb1∆</i> (right). Microscopy and quantification are shown in Fig 7A and 7C and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005390#pgen.1005390.s007" target="_blank">S7 Fig</a>. In <i>YPT1</i> cells, whereas >60% of Hmg1 and Sec61 (green) co-localize with Snc1-PEM in strains defective in vacuolar proteolysis, Sec12, Sec13 and Kar2 are not changed significantly (blue and grey). Hmg1 and Sec61 (green) co-localize with Snc1-PEM in >70% of <i>ypt1-1</i> and >80% of <i>ypt1-1 pep4∆ prb1∆</i> mutant cells, whereas Sec13 and Kar2 (blue and grey) do so in <15% of the cells. The level of Sec12 co-localization with Snc1-PEM is intermediate, ~38% in <i>ypt1-1</i> and <i>ypt1-1 pep4∆ prb1∆</i>. Error bars represent STDEV. Results in panels A and B represent at least two independent experiments.</p

Depletion of Atg9 and Atg2, but not Atg18, result in a macro-ER-phagy phenotype different from that of other core Atgs, and <i>atg9∆</i> is epistatic to <i>atg11∆</i>.

<p><b>A-C</b>. While the level of overexpressed GFP-Snc1-PEM is increased in atg9∆ and atg2∆, but not atg18∆, mutant cells, it does not accumulate in aberrant structures and does not induce UPR. Wild type (WT), <i>atg9∆</i>, <i>atg18∆</i>, and <i>atg2∆</i> mutant cells overexpressing GFP-Snc1-PEM were analyzed as described for <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005390#pgen.1005390.g001" target="_blank">Fig 1A–1C</a>, respectively. The tested phenotypes: the level of GFP-Snc1-PEM protein (<b>A</b>), accumulation of GFP-Snc1-PEM in aberrant structures (<b>B</b>), and induction of the UPR response (<b>C,</b><i>atg1∆</i> is shown as a positive control). <b>B.</b> Shown from top to bottom: DIC, GFP, and % cells with intracellular Snc1-PEM structures. <b>D-G.</b> Atg9 is epistatic to Atg11 in macro-ER-phagy. <i>ATG9</i> was deleted in wild type and <i>atg11∆</i> mutant cells and the effects of overexpression of GFP-Snc1-PEM were determined in the single and double mutants as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005390#pgen.1005390.g001" target="_blank">Fig 1</a> legend. <b>D.</b> Deletion of <i>ATG9</i> in wild type (WT) or <i>atg11∆</i> mutant cells results in an increase of GFP-Snc1-PEM protein level similar to the increase in <i>atg11∆</i> mutant cells. <b>E.</b> Deletion of <i>ATG9</i> in wild type or <i>atg11∆</i> mutant cells results in an increase of intracellular GFP-Snc1-PEM fluorescence. However, only ~20% of the <i>atg9∆</i> single-, and <i>atg9∆ atg11∆</i> double-mutant cells accumulate GFP-Snc1-PEM in aberrant structures, as compared with ~75% of <i>atg11∆</i> mutant cells. Shown from top to bottom: DIC, GFP, % cells with aberrant intracellular GFP-Snc1-PEM structures, ratio of GFP-Snc1-PEM fluorescence inside/PM (30 cells were analyzed for each strain). <b>F.</b> UPR is induced in <i>atg11∆</i>, but not in <i>atg9∆</i> single- and <i>atg9∆ atg11∆</i> double-mutant cells overexpressing GFP-Snc1-PEM. <b>G.</b> UPR can be induced in <i>atg9∆</i> single-, and <i>atg9 atg11∆</i> double-mutant cells overexpressing GFP-Snc1-PEM by tunicamycin. +/- and error bars represent STDEV. Results in this figure represent at least two independent experiments.</p

Snq2-yEGFP is another macro-ER-phagy cargo and its co-overexpression with DsRed-Snc1-PEM has a synergistic effect.

<p><b>A-C.</b> Overexpressed Snq2-GFP accumulates in the ER and induces UPR in <i>ypt1-1</i> mutant cells. Snq2-yEGFP was overexpressed in WT and <i>ypt1-1</i> mutant cells and the following effects were analyzed as described for <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005390#pgen.1005390.g001" target="_blank">Fig 1A–1C</a>, respectively: increase in the level of Snq2-yEGFP protein (<b>A</b>), accumulation of Snq2-yEGFP in aberrant ER structures (<b>B</b>), and induction of the UPR response (<b>C,</b> GFP-Snc1-PEM is shown for comparison). <b>B.</b> The ER marker Sec61 was tagged with mCherry at its C-terminus in WT and <i>ypt1-1</i> mutant cells. Shown from left to right: DIC, GFP, mCherry, Merge, % cells with intracellular Snq2 and % cells in which Snq2 co-localized with Sec61. <b>D-F.</b> Synergistic effect of co-overexpression of Snq2-GFP with DsRed-Snc1-PEM in <i>atg11∆</i> mutant cells. WT (left) and <i>atg11∆</i> mutant cells (right) were transformed with plasmids for overexpression of Snq2, Snc1-PEM or both Snq2 and Snc1-PEM. <b>D.</b> Immunoblot analysis and quantification. Shown from top to bottom: Snq2-yEGFP (using anti-GFP antibodies), Ds-Red-Snc1-PEM (using anti-Snc1 antibodies), G6PDH (loading control), and a bar graph showing fold increase of Snq2-yEGFP (green) and Ds-Red-Snc1-PEM (red) in <i>atg11∆</i> mutant cells over WT. <b>E.</b> Accumulation of macro-ER-phagy cargos in aberrant intracellular structures using live-cells microscopy. Shown from left to right: DIC, GFP, DsRed and Merge. Shown from top to bottom: Snq2, Snc1-PEM, Snq2+Snc1-PEM, and % cells with co-localization (relevant only for co-overexpression) (% cells with ER-phagy cargo accumulation in aberrant intracellular structures is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005390#pgen.1005390.s005" target="_blank">S5A Fig</a>). <b>F.</b> Fluorescence level of intracellular structures (ratio over PM) in <i>atg11∆</i> mutant cells. The bar graph shows Snq2-yEGFP (green) and Ds-Red-Snc1-PEM (red) fluorescence (20 cells were analyzed for each strain). When co-overexpressed in <i>atg11∆</i> mutant cells, the fluorescence level of either protein accumulating in aberrant structures is ~5 fold higher than when overexpressed individually. +/- and error bars represent STDEV. Results in this figure represent at least two independent experiments.</p

Atg9 is epistatic to Ypt1 in macro-ER-phagy and not to the ER-exit regulator Sec12, which is not defective in this process.

<p><b>A-C.</b><i>ATG9</i> was deleted in <i>ypt1-1</i>, WT and <i>sec12ts</i> mutant cells and the effect of overexpression of GFP-Snc1-PEM was determined in single and double mutant cells. Experiments were performed as described for <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005390#pgen.1005390.g001" target="_blank">Fig 1A–1C</a>, respectively. <b>A.</b> Snc1-PEM is increased ~3 fold in <i>atg9∆</i> and <i>sec12ts</i> as compared to ~20 fold in <i>ypt1-1</i> mutant cells. Importantly, <i>atg9∆</i> is epstatic to the <i>ypt1-1</i>, but not to the <i>sec12ts</i>, mutation. Left, immuno-blot analysis, increase of the protein level in mutant versus the WT cells is shown under the blot; right, a bar graph summarizing the quantified data. <b>B.</b> Whereas deletion of <i>ATG9</i> in <i>ypt1-1</i> mutant cells results in three fold lower accumulation of GFP-Snc1-PEM in aberrant structures (85% to 27%), its deletion in <i>sec12ts</i> mutant cells results in two fold increased accumulation (~40% to ~80%). Shown from top to bottom: DIC, GFP, and % cells with intracellular Snc1-PEM structures. <b>C.</b> Deletion of <i>ATG9</i> in <i>ypt1-1</i> mutant cells overexpressing Snc1-PEM results in ~3.5 lower UPR (p-value<0.0005), but a slightly increased UPR in <i>sec12ts</i> mutant cells (p-value = 0.05). <b>D.</b> Atg9 is present on aberrant ER structures that accumulate in <i>ypt1-1</i> mutant cells. WT and <i>ypt1-1</i> mutant cells expressing Atg9-mCherry from the chromosome and GFP-Snc1-PEM from a 2μ plasmid were analyzed by live-cell microscopy. Whereas in WT cells (top), the GFP-Snc1-PEM localizes to the cell membrane and Atg9-mCherry to intracellular puncta, the two proteins co-localize in 100% of the <i>ypt1-1</i> mutant cells (bottom) that accumulate intracellular GFP-Snc1-PEM structures (~80%). Shown from left to right: DIC, GFP, mCherry, merge, % cells with intracellular Snc1-PEM, and % cells with co-localization (number of cells with observed phenotype / total number of cells analyzed). <b>E.</b> GFP-Snc1-PEM is delivered to the vacuole for degradation in <i>sec12ts</i> mutant cells. Accumulation of GFP-Snc1-PEM in vacuoles of <i>sec12ts</i> mutant cells, with and without deletion of the vacuolar protease Pep4, was determined using the FM4-64 dye, which labels the vacuolar membrane. Deletion of <i>PEP4</i> in <i>sec12ts</i> mutant cells results in an increase percent of cells in which GFP-Snc1-PEM accumulates inside the vacuole (from 8% to 100%). Shown from left to right: DIC, GFP, FM4-64 (vacuolar membrane), merge, % cells with aberrant GFP-Snc1-PEM structures, % cells with GFP-Snc1-PEM outside vacuole, and % cells with GFP-Snc1-PEM inside the vacuole. +/- and error bars represent STDEV. Results in this figure represent at least two independent experiments.</p

Characterization of constitutive ER-phagy of excess membrane proteins.

Thirty percent of all cellular proteins are inserted into the endoplasmic reticulum (ER), which spans throughout the cytoplasm. Two well-established stress-induced pathways ensure quality control (QC) at the ER: ER-phagy and ER-associated degradation (ERAD), which shuttle cargo for degradation to the lysosome and proteasome, respectively. In contrast, not much is known about constitutive ER-phagy. We have previously reported that excess of integral-membrane proteins is delivered from the ER to the lysosome via autophagy during normal growth of yeast cells. Whereas endogenously expressed ER resident proteins serve as cargos at a basal level, this level can be induced by overexpression of membrane proteins that are not ER residents. Here, we characterize this pathway as constitutive ER-phagy. Constitutive and stress-induced ER-phagy share the basic macro-autophagy machinery including the conserved Atgs and Ypt1 GTPase. However, induction of stress-induced autophagy is not needed for constitutive ER-phagy to occur. Moreover, the selective receptors needed for starvation-induced ER-phagy, Atg39 and Atg40, are not required for constitutive ER-phagy and neither these receptors nor their cargos are delivered through it to the vacuole. As for ERAD, while constitutive ER-phagy recognizes cargo different from that recognized by ERAD, these two ER-QC pathways can partially substitute for each other. Because accumulation of membrane proteins is associated with disease, and constitutive ER-phagy players are conserved from yeast to mammalian cells, this process could be critical for human health