(a) Co-localization of Rubicon-EGFP-associated structures (green) with the late endosome/lysosome marker Lamp1 (red) (arrows) in the HeLa cells that were transfected with Rubicon-EGFP. Note that some of the Rubicon-EGFP-associated structures displayed “ring”-shape (yellow arrows). Scale bar: 10 μm.
(b) Partial co-localization of Rubicon-EGFP-associated structures (green) with the MVB marker LBPA (red) (arrows) in the HeLa cells that were transfected with Rubicon-EGFP. Scale bar: 10 μm.
(c) Representative ultrastructural images show aberrant expansion of late endosomes/lysosomes structures in the HEK 293T cells over-expressing Rubicon-EGFP. These abnormal organelles are large in size and contained high (orange arrows) or low (black arrows) electron density. Some enclose small vesicles (purple arrows) and some resemble MVB (blue arrows). Scale bars: 500 nm.
(d) Representative ultrastructural images showe late endosome/lysosome-like structures that are labeled by anti-GFP gold particles (panels 3–4) in the HEK 293T cells transiently transfected with Rubicon-EGFP. These structures are enwrapped by double membranes (panel 4 inset) and co-labeled by anti-GFP (developed by DAB) and anti-Lamp1 (gold enhanced) (panels 5–7). Note that mitochondria are mostly negative for Rubicon-EGFP (panel 4). The negative controls are without antibody (panels 1–2). M – mitochondria. Scale bars: 200 nm.

(a) Rubicon siRNA treatment of the NIH 3T3 cells led to decreased levels of p62 and LC3 II under both normal and nutrient-starvation conditions.
(b) Over-expression of Rubicon resulted in increased levels of p62 under both normal and nutrient-starved conditions in HEK 293 cells either stably expressing (upper panel) or transiently transfected with (lower panel) Rubicon-EGFP. The control cells were either stably expressing or transiently transfected with the EGFP-N3 vector.
(c) Vps34 kinase activity. HEK 293T cells were co-transfected with myc-Vps34-Vps15 and FLAG-Rubicon or FLAG vector, either in the absence or in the presence of Beclin 1-EGFP. Myc-Vps34-Vps15 was immunoprecipitated by anti-myc antibody and used for the in vitro kinase assay. The resulting radioactive PtdIns(3)P was separated by TLC, quantified and normalized against the amount of immunoprecipitated myc-tagged Vps34 as measured by Western blot (upper left panel). The quantified results (right panel) showed that over-expressing Rubicon significantly down-regulated the Vps34 kinase activity to 0.58 fold, but only when without Beclin 1 over-expression (asterisk, p=0.04 using one-tailed Student’s t-test with unequal variances, n=4).
(d) Effect of over-expressing FLAG-Rubicon on autophagosome acidification, as monitored by mCherry-GFP-LC3 fluorescence. HeLa cells were transiently co-transfected with mCherry-GFP-LC3 and FLAG-Rubicon (or control FLAG vector). Cells co-expressing mCherry-GFP-LC3 and control FLAG vector contained many red-only puncta along with yellow (indicating presence of both red and green) puncta, suggesting both autolysosomes and nascent autophagosomes (upper panel). In contrast, cells co-expressing mCherry-GFP-LC3 and FLAG-Rubicon contained primarily yellow puncta, suggesting only nascent autophagosomes (lower panel, white arrows). Notably, some cells, which were co-transfected with mCherry-GFP-LC3 and FLAG-Rubicon but expressed high levels of mCherry-GFP-LC3 and undetectable FLAG-Rubicon, contained many red-only puncta (low panel, yellow arrows).
(e) Quantitation of the results in (d) showed that over-expressing FLAG-Rubicon drastically reduced the percentage of red-only puncta (mCherry-LC3) from 39% in the control FLAG vector-transfected control cells to 2% in the FLAG-Rubicon-transfected cells (asterisk, p=2E-26, one-tailed Student’s t-test with unequal variances, n=30), indicating that over-expression of Rubicon blocks autophagosome acidification or maturation.

(a) Local sequence alignment between the C-terminal cystein-rich domain of Rubicon and FYVE domains of several known FYVE-containing proteins. Rubicon does not possess the key consensus sequences of a typical FYVE domain, i.e., N-terminal WxxD, central R[R/K]HHCR and C-terminal RVC (indicated by red bars).
(b) Co-localization of the PtdIns(3)P-enriched lipid domain marker p40 (phox)-PX-EGFP (green) and Rubicon-AsRed (red) on large punctate structures (arrows) in the co-transfected HeLa cells (upper panels). Upon short treatment with 75 nM wortmannin (another PI-3K inhibitor) for 1 h, while the PtdIns(3)P-enriched lipid domains disappeared, the Rubicon-AsRed-positive structures was maintained (lower panels). Scale bars: 10 μm.
(c) Subcellular localization of transiently transfected Rubicon-EGFP, Rubicon(ΔRUN)-EGFP, Rubicon(ΔC)-EGFP or Rubicon(ΔRUNΔC)-EGFP in HeLa cells. In contrast to punctate Rubicon-EGFP and Rubicon(ΔRUN)-EGFP, Rubicon(ΔC)-EGFP and Rubicon(ΔRUNΔC)-EGFP were dispersed in cytoplasm. Abbreviations: ΔRUN – RUN domain deletion, ΔC – cysteine-rich domain deletion. Scale bars: 10 μm.
(d-e) Absence of full-length Beclin 1 (d) or Beclin 1-CE mutant (e) (red) on the Rubicon-EGFP-positive structures (green) in the HEK 293 cells stably expressing Rubicon-EGFP. These cells were transiently transfected with either (d) Beclin 1-AsRed or (e) FLAG-Beclin 1-CE (i.e., the FLAG-tagged Beclin 1 mutant containing both CCD and ECD that mediate the Beclin 1-Rubicon interaction as shown in Fig. S3h). Scale bars, 10μm.
(f) The formation of the Rubicon-EGFP-positive structures was not affected by siRNA knocking-down of Beclin 1 in the HEK 293 cells stably expressing Rubicon-EGFP.
(g) A hypothetic model for the Beclin 1-Vps34protein complexes and their functions. In this model, a core Beclin 1 complex is composed of Vps34/PI-3K, p150/Vps15, Beclin 1, UVRAG and likely substoichiometric Atg14L (indicated by the tight binding and functional connection between Atg14L and Beclin 1). Under physiological conditions, a large Beclin 1-Vps34 complex is formed, including the core complex and Rubicon. This large complex may be reduced to form smaller complexes such as a Atg14L-Beclin 1-containing complex and a Rubicon-containing complex. These smaller complexes are likely the functional units participating in autophagy regulation through modulating the Vps34 lipid kinase activity.

(a) Western blot analysis showed the replacement of endogenous Beclin 1 with Beclin 1-EGFP in Becn1^−/−; Becn1-EGFP/+ mice, as detected by anti-Beclin 1 antibody.
(b) Coomassie-stained SDS-PAGE revealed the Beclin 1-interacting proteins immuno-isolated from the brain and liver tissues of the “rescued” mice (lanes 2 and 4) and of the control Becn1^+/− littermates (lanes 1 and 3), using anti-GFP antibody. Proteins in the gel bands were extracted and identified by mass spectrometry as Vps15/p150 (band 1), Vps34/class III PI-3K (band 3), UVRAG (band 4), Beclin 1-EGFP (band 5) and two novel proteins Atg14L (band 6, asterisk, gi|27369860) and Rubicon (band 2, asterisk, gi|45708948). Notably, UVRAG levels varied with different affinity purification conditions, suggest relatively unstable association of UVRAG with the complex.
(c) Schematic diagrams of the domain structures of Atg14L and Rubicon. Atg14L contains two coiled-coil domains (CCD1 and CCD2), which are also homologous to the SMC domain (Structural Maintenance of Chromosomes). Rubicon contains an N-terminal RUN (for RPIP8, UNC-14 and NESCA) domain, a C-terminal cysteine-rich domain, and a central CCD domain.
(d) Western blot analysis of Atg14L, Rubicon, Vps34 and Beclin 1 in the gel filtration fractions from wild type mouse liver extract showed co-elution of these proteins in fractions 38–45. Atg14L was also eluted in later fractions 51–56. The fractions for the peak elution of thyroglobulin (670 kDa) and γ-globulin (158 kDa) are labeled by arrows. The control siRNA-transfected NIH 3T3 cell lysate was loaded as a positive control (labeled with “1”) for the migration position of the Atg14L protein on SDS-PAGE; the Atg14L siRNA-transfected NIH 3TC cell lysate was loaded as a negative control (labeled with “2”).
(e-f) Co-immunoprecipitation confirmed protein-protein interaction between Atg14L and Rubicon. HEK 293 cells were co-transfected with Atg14L-EGFP and FLAG-Rubicon (e), or Rubicon-EGFP and FLAG-Atg14L (f). Cell lysate was used for immunoprecipitation with anti-GFP antibody, and the resulting immunoprecipitates were blotted with anti-FLAG antibody. Our results showed immunoprecipiration of Rubicon by Atg14L (e) and vice versa (f). WCL: whole cell lysate; IP: immunoprecipirated.

(a) Beclin 1 or Atg14L siRNA reduced Atg14L levels and increased p62/SQSTM1 and LC3 II levels under both normal and nutrient-starvation conditions in NIH 3T3 cells.
(b) Atg14L siRNA, as compared to control siRNA, decreased long-lived protein degradation in NIH 3T3 cells under both normal (p=0.007) and starvation (p=5E-6) conditions (asterisks, one-tailed Student’s t-test with equal variances, n = 4). This difference was diminished when the starved cells were treated with 3-methyladenine (3MA), a PI-3K inhibitor.
(c) Vps34 kinase assay. HEK 293T cells were co-transfected with myc-Vps34-Vps15 and FLAG-Atg14L or FLAG vector, either in the absence or in the presence of Beclin 1-EGFP. Myc-Vps34-Vps15 was immunoprecipitated by anti-myc antibody for the in vitro kinase assay. The resulting radioactive PtdIns(3)P was separated by thin layer chromatography (TLC) (lower left panel), quantified and normalized against the amount of immunoprecipitated myc-tagged Vps34 as measured by Western blot (upper left panel). The quantified results (right panel) showed that over-expressing Atg14L significantly up-regulated the Vps34 kinase activity by 2.5 folds, but only when Beclin 1 was also over-expressed (asterisk, p=0.04, one-tailed Student’s t-test with unequal variances, n=5).
(d) Co-localization of co-expressed Atg14L-EGFP (green) & Beclin 1-AsRed (red) in punctate structures in transiently transfected HeLa cells. Scale bar: 10 μm.
(e) Electron microscopic images show large structures (blue asterisks) that are often enwrapped with double membranes in the HEK 293T cells co-transfected with Atg14L-EGFP and Beclin 1-AsRed: (e1) concentric membrane “rings”; (e2) two large structures (3–5 μm in diameter) contain materials with high electron density; inset, enwrapping double membranes; (e3) numerous autophagosomes (blue arrows) in cytoplasm; (e4) immuno-electron microscopic image of a Atg14L-Beclin 1 structure (labeled with anti-GFP antibody and developed by DAB) enwrapped with concentric membrane “rings”. Abbreviations: M – mitochondria, N – nucleus. Scale bar: 500 nm.
(f-g) EGFP-Atg12 (f) or EGFP-Atg5 (g) (green) was colocalized with the large structures (arrows) that were labeled by Atg14L-AsRed (red) and Beclin 1-myc (blue) in transfected HeLa cells. Some of these structures appeared to be “ring”-shaped (yellow arrows & inset). Scale bar: 10 μm.