QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 3 August 2004
doi: 10.1242/jcs.01287

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Simonsen, A. Articles by Stenmark, H. Search for Related Content PubMed PubMed Citation Articles by Simonsen, A. Articles by Stenmark, H. Social Bookmarking                         What's this?

Alfy, a novel FYVE-domain-containing protein associated with protein granules and autophagic membranes

Anne Simonsen^1,, Hanne C. G. Birkeland^1,, David J. Gillooly¹, Noboru Mizushima^2,3,*, Akiko Kuma^2,3, Tamotsu Yoshimori^4,5, Thomas Slagsvold¹, Andreas Brech¹ and Harald Stenmark^1,

¹ Department of Biochemistry, The Norwegian Radium Hospital, Montebello, Oslo, 0310, Norway
² Department of Cell Biology, National Institute for Basic Biology, 38 Nishigonaka, Myodaijicho, Okazaki 444-8585, Japan
³ PRESTO, Japan Science and Technology Agency, Kawaguchi Center Building, 4-1-8, Honcho, Kawaguchi-shi, Saitama 332-0012, Japan
⁴ CREST, Japan Science and Technology Agency, Kawaguchi Center Building, 4-1-8, Honcho, Kawaguchi-shi, Saitama 332-0012, Japan
⁵ Department of Cell Genetics, National Institute of Genetics, Yata 1111 Mishima, Shizuoka-ken, 411-8540, Japan

View larger version (12K): [in a new window]   Fig. 1. Alfy, a novel human FYVE-domain protein with putative homologues in several species. The presence of known domains in human Alfy and putative Alfy homologues from different species are indicated. Boxes represent conserved regions, whereas dashed lines represent non-conserved regions. The Alfy sequence has been deposited in the EMBL database with accession number AF538685. Putative homologues of Alfy can be found in Drosophila melanogaster (BCHS, AE003611), Caenorhabditis elegans (contig of Z99169+AL032675+Z93390), Dictyostelium discoideum (LvsA, AF088979), Arabidopsis thaliana (AC002330) and Schizosaccharomyces pombe (CAA20312). The mammalian LYST/CHS/Beige protein is also indicated.

View larger version (36K): [in a new window]   Fig. 2. Characterization of anti-Alfy antibodies. (A-D) HeLa cells were transfected with Myc-Alfy2587-3527 and analysed by western blotting (A) or immunofluorescence microscopy with anti-Myc (A,B) and anti-Alfy (A,C) antibodies. (E) The specificity of the anti-Alfy antibody was analysed using affinity-purified anti-Alfy serum or serum depleted of anti-Alfy antibody for immunoblotting of a gel containing cytosol (C) or membrane (M) fractions from HeLa cells. (F) HeLa cells were incubated with an Alfy-specific siRNA, a scrambled siRNA or transfection reagent alone. Equal amounts (60 µg protein) of cell lysates were analysed by western blotting with affinity-purified anti-Alfy antibodies.

View larger version (56K): [in a new window]   Fig. 3. In order to investigate whether Alfy is a peripheral-membrane protein, PNS from HeLa cells was treated with 0.1 M Na2CO3, pH 11, and the resulting cytosol (C) and membrane (M) fractions were subjected to 6% SDS-PAGE before immunoblotting using anti-Alfy antibodies.

View larger version (18K): [in a new window]   Fig. 4. Alfy is recruited to cytosolic structures upon starvation. HeLa cells were starved for 0 minutes (A) or 45 minutes (B-D) and thereafter stained with a rabbit anti-Alfy antibody. Scale bar, 10 µm. (E) HeLa cells were starved for different amounts of time in HBSS and the proportion of cells with Alfy-positive structure was calculated based on confocal immunofluorescence using anti-Alfy antibody. Each point represents the mean of three independent experiments. The error bars show s.e.m. P*<0.03 as determined with the 2 test using the SPSS program version 11 (SPSS, Chicago, IL).

View larger version (47K): [in a new window]   Fig. 5. Alfy is ubiquitously expressed. Extracts from cell lines (A) or mouse tissues (B) were subjected to 6% SDS-PAGE and immunoblotted using rabbit anti-Alfy antibodies. The asterisk in (A) indicates an unidentified protein in cell lines recognized by the anti-Alfy antibody. Abbreviations: B, brain; H, heart; K, kidney; L, lung; M, skeletal muscle; P, pancreas; S, spleen.

View larger version (109K): [in a new window]   Fig. 6. Alfy colocalizes with hAtg5 and LC3 by confocal microscopy. HeLa cells grown on coverslips were starved for 45 minutes in HBSS, permeabilized, fixed and double stained against endogenous Alfy (A,D,G,J) and endogenous EEA1 (B), endogenous LAMP-1 (E), overexpressed GFP-hAtg5 (H) or GFP-LC3 (K). Yellow colour in the merged images (C,F,I,L) indicates colocalization. Colocalization between Alfy and autophagic markers is indicated by arrows. Scale bar, 10 µm.

View larger version (13K): [in a new window]   Fig. 7. The effect of vinblastine on cytoplasmic Alfy-positive vesicles. HeLa cells on coverslips were either left untreated or starved for 2 hours. When indicated, the cells were pretreated with 50 µM vinblastine. Fixed cells were stained with anti-Alfy antibodies and studied by confocal microscopy. Error bars indicate s.e.m. from five experiments.

View larger version (31K): [in a new window]   Fig. 8. Alfy binds PtdIns(3)P in vitro and colocalizes partially with PtdIns(3)P in vivo. (A) Cytosol from HeLa cells was incubated with liposomes containing an equal mixture of phosphatidylserine and phosphatidylinositol, in the presence [PtdIns(3)P] or absence (control) of 5% PtdIns(3)P, included at the expense of phosphatidylinositol. Liposomes were collected by centrifugation and the cytosolic supernatant (S, black) and the liposome pellet (P, grey) were analysed for the presence of Alfy by SDS-PAGE (7.5%) and immunoblotting using the anti-Alfy antibody. Error bars denote s.e.m. from three experiments. (B) Nitrocellulose strips containing the indicated lipids were incubated with GST, GST fused with a tandem FYVE domain from Hrs or GST fused with the FYVE domain of Alfy, and analysed for protein binding. The lipids spotted in column 1 in each strip are indicated to the left, whereas lipids spotted in column 2 are indicated to the right. (C) HeLa cells grown on coverslips were starved for 45 minutes in HBSS, permeabilized, fixed and stained against endogenous Alfy (left), and the presence of PtdIns(3)P detected with GST-2xFYVE-Alexa 488 (middle). The merged image (right) shows that some of the Alfy-positive structures (arrows and inset) colocalized partially with PtdIns(3)P-containing areas in the cells. Scale bar, 10 µm.

View larger version (159K): [in a new window]   Fig. 9. Alfy localizes to a novel filamentous structure. (A) HeLa cells grown on gridded coverslips were starved in HBSS for 45 minutes, fixed and labelled with anti-Alfy to detect cells with Alfy-positive structures (A,B, arrowheads). The same cells were thereafter localized on EM using the grid on the coverslip (B). The Alfy-positive structures detected by immunofluorescence (A) could also be detected at the EM level (B), making it possible to study these structures at a higher resolution (D corresponds to structure 2 and C corresponds to structure 1). This showed that Alfy localizes to a novel filamentous structure (C,D, arrows) that, in some cases, localized close to rough ER membranes (C,D, arrowheads). Occasionally, we observed double-membrane structures embedded within the Alfy-positive structures (E, arrows). The filamentous nature of Alfy structures becomes more evident at a higher magnification (F). Abbreviation: Nu, nucleus.

View larger version (91K): [in a new window]   Fig. 10. Cytoplasmic Alfy-positive structures partially colocalize with ubiquitin-containing profiles and are induced by a proteasome inhibitor. HeLa cells were treated with 50 µM PSI or left alone for 5 hours and then further chased for 18 hours without PSI in the medium. Cells were then double labelled against Alfy (red) and ubiquitin (antibody against mono- and polyubiquitin, FK2; green). In both control cells (A,B) and treated cells (C,D), we observed an apparent colocalization between Alfy and ubiquitin. The number of labelled structures had dramatically increased in the PSI-treated cells (C,D). A closer observation of Alfy-positive structures at high magnification revealed that the markers did not completely colocalize. It was especially evident that some Alfy labelling seemed to extend beyond the ubiquitin-labelled area (E, arrowheads).

View larger version (208K): [in a new window]   Fig. 11. Alfy-like structures are found inside autophagic structures. In order to understand the morphology of Alfy structures more clearly, we treated cells with 50 µM PSI (5-hour pulse followed by 18-hour chase) and prepared them by conventional plastic embedding for EM. We observed filamentous meshworks resembling Alfy-positive structures (asterisk in all micrographs) close to (A, double arrow) or seemingly engulfed by double-membrane structures (B, double arrow). These membranes probably represent ER membranes, as indicated by their continuity with the nuclear envelope (A, arrowheads). The double membranes could form structures resembling autophagosomes (C, double arrow). A more autolysosome-like vesicle with only one limiting membrane (arrowhead) is seen in (D). Abbreviations: Aut, autophagosome; Nu, nucleus.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter