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Feeding-fasting dependent recruitment of membrane microdomain proteins to lipid droplets purified from the liver.

ABSTRACT: Lipid droplets (LDs) are cellular stores of neutral fat that facilitate lipid and protein trafficking in response to metabolic cues. Unlike other vesicles, the phospholipid membrane on the LD is a monolayer. Interestingly, this monolayer membrane has free cholesterol, and may therefore contain lipid microdomains that serve as a platform for assembling proteins involved in signal transduction, cell polarity, pathogen entry etc. In support of this, cell culture studies have detected microdomain-associated "raftophilic" proteins on LDs. However, the physiological significance of this observation has been unclear. Here we show that two proteins (Flotillin-1 and SNAP23) that bind to membrane microdomains associate differently with LDs purified from rat liver depending on the feeding/fasting state of the animal. Flotillin-1 increases on LDs in the fed state, possibly because LDs interact with the endoplasmic reticulum (ER), facilitating supply of flotillin-1 from ER to LDs. Interestingly, this increase in flotillin-1 is correlated with an increase in free cholesterol on the LDs in fed state. In opposite behaviour to flotillin-1, SNAP23 increases on LDs in the fasted state and this appears to mediate LD-mitochondria interactions. Such LD-mitochondria interactions may provide fatty acids to mitochondria for promoting beta-oxidation in hepatocytes in response to fasting. Our work brings out physiologically relevant aspects of lipid droplet biology that are different from, and may not be entirely possible to replicate and study in cell culture.

SUBMITTER: Sadh K

PROVIDER: S-EPMC5553754 | biostudies-other | 2017

REPOSITORIES: biostudies-other

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Project description:Mycobacterium tuberculosis is a global pathogen of significant medical importance. A key aspect of its life cycle is the ability to enter into an altered physiological state of nonreplicating persistence during latency and resist elimination by the host immune system. One mechanism by which M. tuberculosis facilitates its survival during latency is by producing and metabolizing intracytoplasmic lipid droplets (LDs). LDs are quasi-organelles consisting of a neutral lipid core such as triacylglycerol surrounded by a phospholipid monolayer and proteins. We previously reported that PspA (phage shock protein A) associates with LDs produced in Mycobacterium In particular, the loss or overproduction of PspA alters LD homeostasis in Mycobacterium smegmatis and attenuates the survival of M. tuberculosis during nonreplicating persistence. Here, M. tuberculosis PspA (PspAMtb) and a ΔpspA M. smegmatis mutant were used as model systems to investigate the mechanism by which PspA associates with LDs and determine if other Mycobacterium proteins associate with LDs using a mechanism similar to that for PspA. Through this work, we established that the amphipathic helix present in the first α-helical domain (H1) of PspA is both necessary and sufficient for the targeting of this protein to LDs. Furthermore, we identified other Mycobacterium proteins that also possess amphipathic helices similar to PspA H1, including a subset that localize to LDs. Altogether, our results indicate that amphipathic helices may be an important mechanism by which proteins target LDs in prokaryotes.IMPORTANCEMycobacterium spp. are one of the few prokaryotes known to produce lipid droplets (LDs), and their production has been linked to aspects of persistent infection by M. tuberculosis Unfortunately, little is known about LD production in these organisms, including how LDs are formed, their function, or the identity of proteins that associate with them. In this study, an established M. tuberculosis LD protein and a surrogate Mycobacterium host were used as model systems to study the interactions between proteins and LDs in bacteria. Through these studies, we identified a commonly occurring protein motif that is able to facilitate the association of proteins to LDs in prokaryotes.

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Feeding-fasting dependent recruitment of membrane microdomain proteins to lipid droplets purified from the liver.

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