Yun-shu Ying

A Role for Caveolin in Transport of Cholesterol from Endoplasmic Reticulum to Plasma Membrane

Caveolin is a 22-kDa membrane protein found associated with a coat material decorating the inner membrane surface of caveolae. A remarkable feature of this protein is its ability to migrate from caveolae directly to the endoplasmic reticulum (ER) when membrane cholesterol is oxidized. We now present evidence caveolin is involved in transporting newly synthesized cholesterol from the ER directly to caveolae. MA104 cells and normal human fibroblasts transported new cholesterol to caveolae with a half-time of ∼10 min. The cholesterol then rapidly flowed from caveolae to non-caveolae membrane. Cholesterol moved out of caveolae even when the supply of fresh cholesterol from the ER was interrupted. Treatment of cells with 10 μg/ml progesterone blocked cholesterol movement from ER to caveolae. Simultaneously, caveolin accumulated in the lumen of the ER, suggesting cholesterol transport is linked to caveolin movement. Caveolae fractions from cells expressing caveolin were enriched in cholesterol 3-4-fold, while the same fractions from cells lacking caveolin were not enriched. Cholesterol transport to the cell surface was nearly 4 times more rapid in cells expressing caveolin than in matched cells lacking caveolin.

Characterization of a cytosolic heat-shock protein-caveolin chaperone complex. INVOLVEMENT IN CHOLESTEROL TRAFFICKING.

Abstract Caveolin is a 22-kDa protein that appears to play a critical role in regulating the cholesterol concentration of caveolae. Even though caveolin is thought to be a membrane protein, several reports suggest that this peculiar protein can traffic independently of membrane vesicles. We now present evidence that a cytosolic pool of caveolin is part of a heat-shock protein-immunophilin chaperone complex consisting of caveolin, heat-shock protein 56, cyclophilin 40, cyclophilin A, and cholesterol. Treatment of NIH 3T3 cells with 1 μm cyclosporin A or 100 nmrapamycin disrupted the putative transport complex and prevented rapid (10–20 min) transport of cholesterol to caveolae. The lymphoid cell line, L1210-JF, does not express caveolin, does not form an immunophilin-caveolin complex, and does not transport newly synthesized cholesterol to caveolae. Transfection of caveolin cDNA into L1210-JF cells allowed the assembly of a transport complex identical to that found in NIH 3T3 cells. In addition, newly synthesized cholesterol in transfected cells was rapidly (10–20 min) and specifically transported to caveolae. These data strongly suggest that a caveolin-chaperone complex is a mechanism by which newly synthesized cholesterol is transported from the endoplasmic reticulum through the cytoplasm to caveolae.

Synaptotagmin regulation of coated pit assembly.

Synaptotagmins bind clathrin AP-2 with high affinity via their second C2 domain, which indicates they are involved in coated pit function. We now report that expression of synaptotagmins lacking either the second C2 domain or the entire cytoplasmic region potently inhibit endocytosis. Inhibition was dependent on two intramembrane cysteine residues that were found to be essential for synaptotagmin oligomerization. Cells expressing the wild-type, but not the mutant, truncated synaptotagmin fragment had a reduced number of clathrin-coated pits. These results suggest that the formation of synaptotagmin multimers is an important step in the regulation of coated pit assembly.

Caveolin-1 secreting LNCaP cells induce tumor growth of caveolin-1 negative LNCaP cells in vivo.

Caveolin‐1 (Cav‐1) was originally identified as a structural protein of caveolae, which is a plasma membrane domain that regulates a variety of signaling pathways involved in cell growth and migration. Here, we show that expression of Cav‐1 in the Cav‐1‐deficient human prostate cancer cell line LNCaP both stimulates cell proliferation and promotes tumor growth in nude mice. Unexpectedly, Cav‐1 expressing LNCaP (LNCaPCav‐1) cells injected into one side of a nude mouse promoted tumor growth of Cav‐1 negative LNCaP cells injected on the contralateral side of the same animal. The LNCaP tumors were positive for Cav‐1, however, this signal was not caused by migrated LNCaPCav‐1 cells, but we show that this Cav‐1 was secreted by the LNCaPCav‐1 tumors. We demonstrate that conditioned media from LNCaPCav‐1 cells contained Cav‐1 that was associated with a lipoprotein particle ranging in size from 15 to 30 nm and a density similar to high density lipoprotein particle. These results suggest that LNCaPCav‐1 cells secreting Cav‐1 particle produce an endocrine factor that stimulates tumor growth.

Rab-regulated membrane traffic between adiposomes and multiple endomembrane systems.

Lipid droplets play a critical role in a variety of metabolic diseases. Numerous proteomic studies have provided detailed information about the protein composition of the droplet, which has revealed that they are functional organelles involved in many cellular processes, including lipid storage and metabolism, membrane traffic, and signal transduction. Thus, the droplet proteome indicates that lipid accumulation is only one of a constellation of organellar functions critical for normal lipid metabolism in the cell. As a result of this new understanding, we suggested the name adiposome for this organelle. The trafficking ability of the adiposome is likely to be very important for lipid uptake, retention, and distribution, as well as membrane biogenesis and lipid signaling. We have taken advantage of the ease of purifying lipid-filled adiposomes to develop a cell-free system for studying adiposome-mediated traffic. Using this approach, we have determined that the interaction between adiposomes and endosomes is dependent on Rab GTPases but is blocked by ATPase. These methods also allowed us to identify multiple proteins that dynamically associate with adiposomes in a nucleotide-dependent manner. An adiposome-endosome interaction in vitro occurs in the absence of cytosolic factors, which simplifies the assay dramatically. This assay will enable researchers to dissect the molecular mechanisms of interaction between these two organelles. This chapter provides a detailed account of the methods developed.