A Critical Role of Cavin (Polymerase I and Transcript Release Factor) in Caveolae Formation and Organization*
- 1 To whom correspondence should be addressed: Boston University School of Medicine, 715 Albany St., K402, Boston, MA 02118. Tel.: 617-638-4044; Fax: 617-638-4208; E-mail: ppilch{at}bu.edu.
Abstract
Cavin (PTRF) has been shown to be a highly abundant protein component of caveolae, but its functional role there is unknown. Here, we confirm that cavin co-localizes with caveolin-1 in adipocytes by confocal microscopy and co-distributes with caveolin-1 in lipid raft fractions by sucrose gradient flotation. However, cavin does not directly associate with caveolin-1 as solubilization of caveolae disrupts their interaction. Cholesterol depletion with β-cyclodextrin causes a significant down-regulation of cavin from plasma membrane lipid raft fractions. Overexpression of cavin in HEK293-Cav-1 cells and knockdown of cavin in 3T3-L1 adipocytes enhances and diminishes caveolin-1 levels, respectively, indicating an important role for cavin in maintaining the level of caveolin-1. A truncated form of cavin, eGFP-cavin-1-322, which lacks 74 amino acids from the C-terminal, reveals a microtubular network localization by confocal microscopy. Disruption of cytoskeletal elements with latrunculin B or nocodazole diminishes cavin expression without affecting the caveolin-1 amount. We propose that the presence of cavin on the inside surface of caveolae stabilizes these structures, probably through interaction with the cytoskeleton, and cavin therefore plays an important role in caveolae formation and organization.
Footnotes
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↵2 The abbreviations used are: PTRF, polymerase I and transcript release factor; Cav-1, caveolin-1; PM, plasma membrane; LM, light microsomes; HM, heavy microsomes; β-CD, β-methylcyclodextrin; NCZ, nocodazole; LatB, latrunculin B; DMEM, Dulbecco's modified Eagle's medium; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline; MES, 4-morpholineethanesulfonic acid; shRNA, short hairpin RNA; IP, immunoprecipitation; eGFP, enhanced green fluorescent protein; HES, HEPES EDTA sucrose.
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↵* This work was supported by National Institutes of Health Grants DK-30425 and 56935 (to P. F. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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- Received September 20, 2007.
- Revision received November 29, 2007.
- The American Society for Biochemistry and Molecular Biology, Inc.
