Fig. 1.
Primary sequences of A. pulchella Rab4 (ApRab4). The start codon and a proceeding in-frame stop codon are in bold letters. The stop codon is indicated by an asterisk. The putative polyadenylation signal is underlined.ApRab14-L full length cDNA
Fig. 2.
Primary sequences of A. pulchella Rab14-L (ApRab14-L). The start codon and a proceeding in-frame stop codon are in bold letters. The stop codon is indicated by an asterisk. The putative polyadenylation signal is underlined.ApRab14-S full length cDNA
GGCCATT 7 ACGGCCGGGAATCACGAGATCCGCCATCTTTCCTAAAGTCTTCTT 52 CCGGATTTC TAATATAATTTAATATTCCCTTACCAGGTCGCAAAG 97 ATGGCAGCGACAGGGCCGTACAATTATTCCTATATATTCAAGTAT 142
M A A T G P Y N Y S Y I F K Y 1 5 ATTATTATAGGTGATATGGGTGTTGGTAAGTCATGTCTCCTTCAC 187 I I I G D M G V G K S C L L H 3 0 CAATTCACAGAAAAGAAATTCATGGCAGACTGCCCTCACACAATT 232 Q F T E K K F M A D C P H T I 4 5 GGTGTAGAGTTTGGCCCAAGAATAATAGAGGTAGCTGGACAAAAG 277 G V E F G P R I I E V A G Q K 6 0 ATCAAGCTTCAGATATGGGATACGGCAGGCTACTATTTATCTCCT 322 I K L Q I W D T A G Y Y L S P 7 5 TAAGCTGAATCAAAATTTACAAGTTAATGAAAAACATATTGAATT 367
*
CT CATTTGATTATAAAC T CCTTGCAGTTAGT CTCTTGTGT CTTCA 4 1 2 CATTGTAGCTAGATTAATGTGATGACACCGGAAATAGCTGCAAGG 457 AGCCTAACTGAACGAAGCTTAACTGTTGTTACAGTAAAATATATT 502 T G CT GT G GT TAAA AT TAC AT T GTAT T C AG G AT T T G AAT T T T T TT T 5 4 7 CAATAAGATAAG ACAG CTCTTCATTTGTTAAAATGGCTGATAAAC 5 92 TAC ACTAAT T GAC AGT GAAT GTAATTT GAATATT GT CATAGC AAC 6 37 CC AG AAT T CTACAT TTCC AT GAC AAC C ACT GNT GT CTATAAC ATT 68 2 GTCATGGTAACTTAGAAGAAGT 704
Fig. 3.
Primary sequences of A. pulchella Rab14-S (ApRab14-S). The start codon and a proceeding in-frame stop codon are in bold letters. The stop codon is indicated by an asterisk. The putative polyadenylation signal is underlined.Fig. 4.
Multi-alignment of ApRab4 protein with selected Rab4 relatives.Amino acid residues identical in all Rab4 proteins are shown on a black background; residues conserved in most members are shown on a gray background. Sequence motifs that are highly conserved among Rab proteins are indicated by solid lines and labeled. Functional motifs involved in
GTP-binding are marked with solid lines and numbered (G1-G4). Sequence motifs that are highly conserved in Rab family proteins are indicated by solid lines and labeled (RabF1–RabF5). The putative C-terminal double cysteine prenylation signal is underlined and labeled with the letter P. The GenBank accession numbers of the selected Rab4 proteins are: AAP97171 (human);
AAL11725 (mouse); AAH44974 (frog); NP 523777 (fly); NP 001004002 (fish); and NP 059051 (rat).
Fig. 5.
Multi-alignment of ApRab14-L protein with selected Rab14 relatives.Amino acid residues identical in all Rab14 proteins are shown on a black background; residues conserved in most members are shown on a gray background. Sequence motifs that are highly conserved among Rab proteins are indicated by solid lines and labeled. Functional motifs involved in
GTP-binding are marked with solid lines and numbered (G1-G4). Sequence motifs that are highly conserved in Rab family proteins are indicated by solid lines and labeled (RabF1–RabF5). The putative C-terminal double cysteine prenylation signal is underlined and labeled with the letter P. The GenBank accession numbers of the selected Rab4 proteins are: NP 057406 (human); NP 080973 (mouse); NP 001007505 (frog); NP 477171 (fly); NP 958903 (fish);
and NP 446041 (rat).
Rab4
rApRab4 Ap rApRab14-L Ap
Fig. 7.
Western blot analysis of ApRab4 and ApRab14-L in Aiptasia protein sample. The rat anti-ApRab4 and anti- ApRab14-L polyclonal antibodies (1:500 dilution) were used in combination with a HRP-conjugated goat anti-mouse IgG secondary antibody (1:10,000 dilution) to detect ApRab4 and ApRab14-L proteins. Ap, 14µg of total Aiptasia protein; rApRab4 and rApRab14-L, affinity-purified recombinant histine-tagged ApRab4 and ApRab14-L proteins as positive control. The recombinat protein contains additional 34 amino acid residues than its parental, WT-type protein. Protein fractions were prepared as described in “Material and Methods”, and equalamount of protein extracts were fractionated in 15% SDS-PAGE gel. Positive protein bands were visualized by chemiluminescence using the Renaissance Western blot reagent from NEN.
ApRab4-WT ApRab4-S22N ApRab4-Q67L
ApRab14-L-WT ApRab14-L-S26N ApRab14-L-Q71L
Fig. 8.
EGFP reporter analyses of ApRab4 and ApRab14-L (wild-type and mutant forms) in HeLa cells. Plasmid encoding either the EGFP-ApRab4, EGFP-ApRab14-L or their mutant forms were introduced into HeLa cells using FuGENE 6. Bars: 10µmApRab4 Transferrin Merge
ApRab14-L Transferrin Merge
Fig. 9.
Co-localization of EGFP-ApRab4 and -ApRab14-L with exogenous transferrin. Plasmids encoding either the EGFP-ApRab4 or EGFP-ApRab14-L fusion proteins were introduced into HeLa cells via FuGENE 6-mediated transfection. Sixteen hours after transfection, cells were incubated with 1µM transferrin-Texas Red for 30 min at 37℃ to label transferring-containing recycling compartments. Fluorescence images were obtained directly from live cells without prior fixation by epi-fluorescence microscopy. Arrows indicate the overlapping signals. Bars: 10µmApRab4 dextran-TRICT (10 min) Merge
ApRab4 dextran-TRICT (30 min) Merge
Fig. 10.
Co-localization of EGFP-ApRab4 with dextran- TRICT (70 kDa).Plasmid encoding the EGFP-ApRab4 fusion protein was introduced into HeLa cells via FuGENE 6-mediated transfection. Sixteen hours after transfection, cells were incubated with dextran-TRICT (70 kDa) for 10 min and 30 min at 37℃ to label early and late stages of endosomes. Fluorescence images were obtained directly from live cells without prior fixation by epi-fluorescence microscopy. Arrows indicate the overlapping fluorescence signals. Bars: 10µm
ApRab14-L dextran-TRICT (10 min) Merge
ApRab14-L dextran-TRICT (30 min) Merge
Fig. 11.
Co-localization of EGFP-ApRab14-L with dextran- TRICT (70 kDa). Plasmid encoding the EGFP-ApRab14-L fusion protein was introduced into HeLa cells via FuGENE 6-mediated transfection. Sixteen hours after transfection, cells were incubated with dextran-TRICT (70 kDa) for 10 min and 30 min at 37℃ to label early and late stages of endosomes. Fluorescence images were obtained directly from live cells without prior fixation by epi-fluorescence microscopy. Arrows indicate the overlapped signals. Bars:10µm
ApRab4 LysoTracker-red Merge
ApRab14-L LysoTracker-red Merge
Fig. 12.
Co-localization of EGFP-ApRab4 and -ApRab14-L with LyoTracker. Plasmid encoding the EGFP-ApRab4 and EGFP-ApRab14-L fusion proteins were introduced into HeLa cells via FuGENE 6-mediated transfection. Sixteen hours after transfection, cells were incubated with 75 nM LysoTracker- red for 15 min at 37℃ to label acidic compartments.Fluorescence images were obtained directly from live cells without prior fixation by epi-fluorescence microscopy. Arrows indicate the non-overlapping signals. Bars: 10µm
ApRab14-L TR-C5-ceramide Merge
ApRab14-L-S26N TR-C5-ceramide Merge
Fig. 11.
ApRab14-L-Q71L TR-C5-ceramide Merge
Fig. 13.
Co-localization of wild-type EGFP-ApRab14 and its mutants with TR-C5-ceramide. Plasmids encoding either the EGFP-ApRab14 or EGFP-ApRab14-L-S26N and ApRab14 -L-Q71L fusion proteins were introduced into HeLa cells via FuGENE 6-mediated transfection. Sixteenhours after transfection, cells were incubated with TR-C5-ceramide for 30 min at 4℃ to label sphingolipids. Fluorescence images were obtained directly from live cells without prior fixation by epi-fluorescence microscopy. Bars:
10µm
ApRab4
ApRab14-L
Fig. 14.
Immunofluorescence analysis of endogenous ApRab4 and ApRab14-L in Aiptasia host cells housing zooxanthellal symbionts. The polyclonal rat anti-ApRab4 and ApRab14-L were used as 1st antibody to label and the Cy3-conjugated goat anti-mouse IgGs was used as 2nd antibody to visualize endogenous ApRab4 and ApRab14-L in zooxanthellae- containingAiptasia cells. ApRab4 and ApRab14-L staining signals were detected on
phagosomes containing lived zooxanthellae. Nucleus was labeled by H33258 staining. Arrows and the letter N indicate the nuclei and the phagosome membrane, respectively. Bars: 10µm
N N
ApRab4
ApRab14-L
Fig. 15.
Intracellular distribution of endogenous ApRab4 and ApRab14-L inAiptasia phagocytes. Identification of Aiptasia phagocytes was aided by the
presence of latex beads (blue fluorescence). Fixed cells were stained for either ApRab4 or ApRab14-L with rat anti-ApRab4 or rat anti-ApRab14-L polyclonal as the primary antibody, a Cy3-conjugated goat anti-mouse IgGs as the secondary antibody. Nucleus was labeled by H33258 staining. Arrows and the letter N in these patterns indicate the phagosomes and the nucleus, respectively. Bars: 10µm
N N
Fig. 16.
Immunofluorescence analysis of endogenous ApRab4 and ApRab14 association with resident zooxanthellae- containing phagosomes (symbiosomes) in DCMU-treated or untreated Aiptasia cells. The rat anti-ApRab4 and rat anti- ApRab14-L IgGs were affinity-purified and used to label, and the Texas Red-conjugated goat anti-rat IgGs was used to visualize endogenous ApRab4 and ApRab14-L in zooxanthellae-containing Aiptasia cells. ApRab4 and ApRab14-L associated with the symbiosomes as a function of the duration of DCMU treatment. ApRab4(□), ApRab5(▲), ApRab7(◇), and ApRab14-L(○)0 10 20 30 40 50 60 70 80 90 100
0 min 30 min 60 min
length of incubation (min) posit
ive c ount s (% )
Fig. 17.
Phagocytosis of latex bead showed the tendency of association of ApRab4 and ApRab14-L with the phagosomes. ApRab4 and ApRab14-L were detected on punctate structures and on latex-bead containing phagosomes in Aiptasia digestive cells. Fixed cells were stained for ApRab4 and ApRab14-L with either the rat anti-ApRab4 or the rat anti-ApRab14-L IgGs as the primary antibody (1:50 dilution), a Texas Red-conjugated goat anti-rat IgGs was used as the secondary antibody (1:1000 dilution). ApRab4 (□), ApRab5 (▲), ApRab7 (◇), and ApRab14 (○)0 10 20 30 40 50 60 70 80 90
15 min 30 min 45 min
length of incubation (min) po sit
iv e co un ts (%
)
Fig. 18.
Phagocytosis of heat-killed zooxanthellae showed the tendency of association of ApRab4 and ApRab14-L with the phagosomes in Aiptasia digestive cells. Fixed cells were stained for ApRab4 and ApRab14-L with either the rat anti-ApRab4 or the rat anti-ApRab14-L IgGs as the primary antibody (1:50 dilution), a Texas Red-conjugated goat anti-rat IgGs was used as the secondary antibody (1:1000 dilution). ApRab4 (□), ApRab5 (▲), ApRab7 (◇), and ApRab14 (○)0 10 20 30 40 50 60 70
15 min 30 min 45 min
length of incubation (min) posit
ive coun ts (%
)