Lipid droplets (LDs) are dynamic lipid stores present in all eukaryotic cells (1
). Efficient assembly and metabolization of LDs into energy probably represents a dominant evolutionary advantage for cells. Excessive and long-term accumulation of LDs is a cellular hallmark of widespread human diseases including obesity, diabetes and arteriosclerosis.
Initially considered to be inert cytoplasmic lipid inclusions, LDs are now understood as complex organelles with a central role in the regulation of cellular lipids, key players in the complex dance of diverse lipids moving through cells. When redistribution of subcellular lipids fails, overall lipid homeostasis is impaired, with severe cellular consequences. Exactly how such lipid distributions are controlled is unclear, but proteomic and other studies suggest significant exchanges of proteins between LDs and a variety of other organelles such as mitochondria and endoplasmic reticulum (ER), potentially facilitating lipid redistribution.
To understand how droplets might exchange proteins and lipids with other cellular compartments, it is important to understand droplets' formation and also how proteins are targeted to them. The prevalent model (1
) suggests that LD biogenesis occurs by esterification and gradual gathering of neutral lipids within the bilayer of the ER. However, some evidence is inconsistent with a model of uncontrolled random gathering: nascent LDs likely originate in specific subdomains within the ER with a unique phospholipid composition (3
); genes encoding enzymes that synthesize phospholipids are determinants of lipid-droplet size and number. Phospholipids details of the monolayer likely represent a first level of regulation of LD function (6
LD formation and lipid mobilization are also likely orchestrated by proteins on the droplets surface (7
). Topologically, there are two structural classes of LD-resident proteins. The first contains proteins without a membrane-integral hydrophobic domain (Hyd), which may be targeted to LDs from the cytosol [see alternative model in (18
)]. This group includes LD-resident proteins such as perilipins (19
), adipose differentiation-related protein (ADRP) (20
) or Rab18 (21
). In contrast, the second group has a Hyd that is most likely inserted in the ER, allowing the protein to laterally diffuse to droplets. This group includes oleosins and caleosins in plants (23
), viral components such as hepatitis C virus (HCV) and GB virus B (GBV-B) core proteins (25
), hairpin membrane-anchored proteins such as caveolins (CAVs) and stomatin (12
), and S-adenosylmethionine (SAM) methyltransferases such as erg6 in Saccharomyces cerevisiae
or ALDI and AAM-B in mammalian cells (11
). This complex protein composition likely confers to the LD, a second level of functional regulation. Indeed, some LD-resident proteins – including CAV – dynamically associate with the organelle upon specific and temporary requirements of the cell (9
). Unfortunately, it is unclear how such dynamic association is controlled: a consensus-targeting motif necessary and sufficient for sorting proteins into LDs has not been demonstrated, and thus the regulation of the LD by a specific and timely targeting of proteins remains largely unexplored.
CAV's dynamic association and regulation is particularly important. Mammalian cells likely use two different organelles to store lipids – intracellular LDs and plasma membrane caveolae (34
) – and thus regulatory circuits to coordinate their functions are critical. Biophysically, caveolae organize as stable flask-shaped ordered domains with a high density of lipids such as glycosphingolipids, sphingomyelin and cholesterol.
CAVs likely provide a critical link between these two storage organelles. They are essential components of caveolae, with a unique capacity to assemble and stabilize caveolae at the plasma membrane. CAVs could also act as sensitive lipid-organizing molecules not only at the cell surface but also on LDs (9
). Importantly, CAVs associate with LDs following particular cell requirements. They thus may regulate LD function under specific conditions, potentially even contributing to transfer of lipids either to, or away from, the droplets.
Consistent with such a role, CAV1 redistributes from the cell surface to LDs upon treatment with cholesterol or fatty acids, and during liver regeneration. Inhibition of caveolae internalization also inhibits cholesterol-stimulated association of CAV1 with LDs (32
). Further, adipocytes from CAV1 gene-disrupted mice show decreased levels of free cholesterol in LDs and CAV1−/−
hepatocytes cannot form LDs during liver regeneration (38
). Consequently, a link between the trafficking of CAV and a lipid sensing and storage function of the protein has been convincingly proposed (34
Additional support for the hypothesis that appropriate control of CAV's localization could contribute to control of cellular lipids comes from our studies with CAV mutants (29
). Unlike normal CAVs with regulated LDs association (see above), the truncation mutant CAVDGV
(N-terminal deletion of 54 residues of CAV3) constitutively accumulates on LDs. Consistent with a role for CAVs in lipid homeostasis, although the mutant protein is irreversibly confined to the droplets, the expression of CAVDGV
promotes a lipid imbalance in other cellular compartments. CAVDGV
causes the accumulation of free cholesterol in late endosomes and the irreversible assembly of neutral lipids in LDs. CAVDGV
also decreases free cholesterol at the cell surface and in the Golgi complex, and the induced perturbation of cellular lipid regulation alters specific signaling pathways at the cell surface such as the H-Ras-mediated activation of Raf-1. The inhibitory effect of CAVDGV
on signaling is completely reversed by replenishing the cell membrane with cholesterol and mimicked by depletion of membrane cholesterol (39
). Therefore, by correct regulation of its localization, it is likely that CAV facilitates multiple cellular processes.
How is CAV's localization controlled? It is clearly essential to identify the molecular determinants involved in the transport of CAVs into the droplets. The prevalent model postulates that the transfer of CAVs into LDs is not regulated, but that simple overaccumulation in the ER diverts the protein into nascent LDs. Proper packing of the central hairpin Hyd is proposed as the only molecular determinant during this process (35
). Certainly, truncation of the Hyd of CAVs, stomatin, oleosin, the HCV core protein and AAM-B completely abolishes their association with LDs (12
), demonstrating that the Hyd is required. However, is the Hyd sufficient for targeting CAV1 into droplets? Increased cholesterol promotes trafficking of CAV from the plasma membrane into LDs (32
), and CAV leaves LDs when fatty acids are removed or at later stages of liver regeneration when LDs are still abundant (36
). Thus, additional levels of regulation appear likely. Multiple lines of evidence suggest that the Hyd alone may not be sufficient. First, similar to CAV, the Hyd of the AAM-B protein was postulated to be sufficient (although with a low efficiency), to partially target proteins into droplets (31
), but this appears incorrect because the same study found that deletion of a short sequence C-terminally flanking the Hyd is sufficient to completely abrogate transport of AAM-B into LDs, although the mutant protein still included an intact Hyd. Second, a green fluorescent protein (GFP)-tagged Hyd of CAV2 does not localize to LDs (27
). Thus, a Hyd alone is likely insufficient to efficiently sort CAVs within the ER into LDs and additional information is required.
Here, we use mutational analysis to investigate the CAVs region(s) necessary and sufficient for sorting to LDs. We show that the Hyd is necessary but not sufficient for targeting CAVs to droplets; instead, targeting of CAVs into droplets requires two cooperative/auxiliary sequences. First, the Hyd anchors CAVs to the ER. Next, CAVs are specifically sorted by positively charged sequences (Pos-Seqs) into LDs. To gain further mechanistic insight into sorting, we identified functionally interchangeable sorting information within ALDI, and use the information to retarget the cytosolic protein GFP to droplets.