Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2004 Sep 14;101(37):13501-6.
doi: 10.1073/pnas.0405664101. Epub 2004 Sep 7.

The inositol polyphosphate 5-phosphatase Ocrl associates with endosomes that are partially coated with clathrin

Alexander Ungewickell  1 , Michael E Ward, Ernst Ungewickell, Philip W Majerus
Affiliations

The inositol polyphosphate 5-phosphatase Ocrl associates with endosomes that are partially coated with clathrin

Alexander Ungewickell et al. Proc Natl Acad Sci U S A. .

Abstract

The subcellular localization of Ocrl, the inositol polyphosphate 5-phosphatase that is mutated in Lowe syndrome, was investigated by fluorescence microscopy. Ocrl was localized to endosomes and Golgi membranes along with clathrin, giantin, the mannose 6-phosphate receptor, transferrin, and the early endosomal antigen 1 endosomal marker in fixed cells. The endosomal localization of Ocrl was confirmed by live-cell time-lapse microscopy in which we monitored the dynamics of Ocrl on endosomes. GST binding assays show that Ocrl interacts with the clathrin terminal domain and the clathrin adaptor protein AP-2. Our findings suggest a role for Ocrl in endosomal receptor trafficking and sorting.

PubMed Disclaimer

Figures

Fig. 1.
Fig. 1.
IF microscopy of Cos7 cells transiently transfected with GFP-Ocrl. Cells in a–c were serum-starved overnight in DMEM/0.1% BSA and serum-stimulated for 5 min before fixation. Cells in b and c were loaded for 30 min with 50 μg/ml Tf-Alexa 555 before serum stimulation. (a) GFP-Ocrl with clathrin and giantin stain. Overlay includes GFP-Ocrl (green), clathrin (red), and giantin (blue). (b) GFP-Ocrl-expressing cells were loaded with Tf-Alexa 555 and stained for clathrin. Overlay includes GFP-Ocrl (green), clathrin (red), and Tf (blue). (c) GFP-Ocrl-expressing cells were loaded with Tf-Alexa 555 and stained for the CI-M6PR. Overlay includes GFP-Ocrl (green), CI-M6PR (red), and Tf (blue). (d) GFP-Ocrl-expressing cells were stained for EEA1. Overlay includes GFP-Ocrl (green) and EEA1 (red).
Fig. 2.
Fig. 2.
GFP-Ocrl before (a) and 23 min after (b) serum stimulation. A large increase in the number of peripheral GFP-Ocrl punctae (solid arrowhead) and rings (open arrowhead) was observed after serum stimulation. Not all vesicular structures contain Ocrl, and some acquire Ocrl upon serum stimulation (open arrowheads) (see Movie 1).
Fig. 3.
Fig. 3.
Proteins associated with Ocrl. (a) Live fluorescence microscopy of CFP-clathrin, GFP-Ocrl, and Tf-Alexa 568 after serum stimulation in Cos7 cells. The three proteins colocalize on the large vesicular structure marked by the white arrow. The open arrowhead points at a black vesicular structure that buds off a larger elongated structure. CFP-clathrin, GFP-Ocrl, and Tf-Alexa 568 are found around this budding vesicle (white arrowhead). The three proteins subsequently move with the vesicle toward the top of the picture (see Movie 3). A stationary vesicular structure is marked by an arrow. (b) Live fluorescence microscopy of CFP-CD-M6PR, GFP-Ocrl, and DsRed-clathrin after serum stimulation. Two structures that contain all three proteins are pointed out at time 0 s. The structure marked by the arrowhead then moves over the next 23 s; the other vesicular structure remains relatively stationary (see Movie 4).
Fig. 4.
Fig. 4.
Ocrl associations. (a) Domain structure of Ocrl. Locations of the FEDNF AP-2-binding motif and the clathrin box motif LIDLE are shown. (b) Interaction between Ocrl and TD. Flag-Ocrl, GFP-Ocrl, and GFP-Ocrl LIAAA were transiently transfected into HeLa cells. GST or GST-TD beads were incubated with HeLa cell lysates. Sixteen percent of bound (P) and 2% of unbound (S) proteins were analyzed by anti-Flag Western blotting. Fourteen percent of bound (P) and 2% of unbound (S) proteins were analyzed by anti-GFP Western blotting. (c) Interaction of purified Ocrl and AP-2α. Ten percent of bound (P) and 1% of unbound (S) fractions of binding assays with GST, GST-α (AP-2α), GST-γ (AP-1γ), and GST-GGA2 (170–613) were analyzed by Ocrl Western blotting. (d) Requirement of FxDxF motif in Ocrl for AP-2 binding. Purified Ocrl or Ocrl AEANF mutant protein produced in SF9 cells was incubated with GST or GST-α appendage subunit of AP-2. Ten percent of bound and 1% of unbound fractions were analyzed by Ocrl Western blotting.
Fig. 5.
Fig. 5.
Ocrl copurifies with CCVs. Fractions were prepared as described by Maycox et al. (13). H, homogenate; S1, postnuclear supernatant; P1, nuclei/intact cells/large cell fragments; S2, supernatant minus synaptosomes; P2, crude synaptosomes; LS1, mostly cytosol; LP1, plasma membrane/coated pits/endosomes; LP2, endosomes/synaptic vesicles; LP3, CCV/remaining synaptic vesicles; CCV, highly purified CCV. The amounts of total protein loaded onto gel were as follows: H, 22.5 μg; S1, 8.25 μg; P1, 40.5 μg; S2, 6.4 μg; P2, 9 μg; LS1, 0.75 μg; LP1, 29.25 μg; LP2, 1.95 μg; LP3, 0.75 μg; CCV, 1.8 μg. The blot was probed for clathrin and Ocrl and then stripped and probed for AP-2, the multivesicular endosome marker hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), and the synaptic vesicle protein synaptotagmin (Syt).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Attree, O., Olivos, I. M., Okabe, I., Bailey, L. C., Nelson, D. L., Lewis, R. A., McInnes, R. R. & Nussbaum, R. L. (1992) Nature 358, 239–242. - PubMed
    1. Nussbaum, R. (2000) in The Metabolic and Molecular Bases of Inherited Disease, eds. Scriver, C. R., Sly, W.S., Childs, B., Beaudet, A. L., Valle, D., Kinzler, K. W. & Vogelstein, B. (McGraw–Hill, New York), 8th Ed., pp. 6257–6266.
    1. Majerus, P. W., Kisseleva, M. V. & Norris, F. A. (1999) J. Biol. Chem. 274, 10669–10672. - PubMed
    1. Zhang, X., Hartz, P. A., Philip, E., Racusen, L. C. & Majerus, P. W. (1998) J. Biol. Chem. 273, 1574–1582. - PubMed
    1. Suchy, S. F., Olivos-Glander, I. M. & Nussbaum, R. L. (1995) Hum. Mol. Genet. 4, 2245–2250. - PubMed

Publication types

MeSH terms