A Critical Role of Cavin (Polymerase I and Transcript Release Factor) in Caveolae Formation and Organization

Libin Liu,Paul F Pilch

doi:10.1074/jbc.m707890200

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 beta-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.

Highlights

In certain cells, morphologically defined caveolae can be formed by the expression of their structural protein caveolin-1
Caveolin-2 appears to require the expression of caveolin-1 to target it to plasma membrane caveolae [24], and it does not seem capable of forming caveolae when expressed on its own
The major protein components of immunoisolated caveolae were identified by mass spectrometry-based sequencing to be caveolin-1 and -2, CD36, semicarbazide-sensitive amine oxidase, and polymerase I and transcript release factor (PTRF)2 [8, 26], known as cavin [27]

Summary

Introduction

Morphologically defined caveolae can be formed by the expression of their structural protein caveolin-1. Our data confirm that cavin co-localizes with caveolin-1 in adipocytes by confocal microscopy and co-distributes with caveolin-1 in lipid raft fractions in sucrose gradients. These data further confirm that cavin is a lipid raft constituent that co-localizes with caveolin-1 in caveolae.

Results

Conclusion