Combined techniques identify proteins specific to lipid droplets in S. cerevisiae

Research Areas:

Lipid droplet proteins, S. cerevisiae, Fluorescence microscopy, Green fluorescent protein, monodansyl pentane

Imaging Needs:

High sensitivity

Imaging System:
  • Nikon ECLIPSE Ti 2000 microscope
  • Yokogawa CSU-X1 spinning disk
  • Proteins tagged with green fluorescent protein
  • LDs stained with monodansyl pentane
  • Excitation: solid-state lasers at 405 nm, 491 nm and 561 nm
  • Hamamatsu ImagEM EM-CCD camera
  • Micro-Manager image acquisition and control software
  • Huygens SVI and Image J image processing software
Imaging cellular events in real time

Find out how Fang Huang, Jeorg Bewersdorf and colleagues use the sCMOS technology in the ORCA-Flash4.0 camera to achieve video-rate imaging at nanometer scales. Read now.

THE QUESTION

How can researchers identify the specific network of biomolecules responsible for the structure and functions of an intracellular organelle?

To fully understand the workings of an intracellular organelle requires knowledge of the biomolecules that carry out its functions—key among them, proteins. Researchers can readily identify the location and abundance of a specific protein, for example using antibodies or gene tagging. However, systematically cataloging the network of proteins important to a given cellular function or organelle is more challenging.

THE BARRIERS

Methods to quantify gene expression—specifically, mRNA—provide valuable knowledge of cellular processes. Unfortunately, mRNA levels do not always correlate well with protein abundance, and cannot provide direct insights into functional modifications and localization of the protein itself. More recent methods incorporating mass spectrometry of cell lysate fractions have made possible a rush of new information about protein composition and dynamics in cells, protein complexes, and organelles.1 Cross-contamination of fractions, however, often results in false positives.

The proteins of lipid droplets in particular have proven difficult to characterize. These hydrophobic organelles play important roles in lipid storage and metabolism in all eukaryotes and even some prokaryotes. Because of their close physical proximity to other organelles, especially the endoplasmic reticulum, lipid droplets can be challenging to purify. In addition, proteins that appear with lipid droplets under confocal microscopy could instead be associated with the membranes of adjacent organelles. As a result, reports of lipid droplet inventories in the literature have been highly variable.

THE SOLUTION

High confidence proteomic analysis of yeast LDs identifies additional droplet proteins and reveals connections to dolichol synthesis and sterol acetylation
Erin Currie, Xiuling Guo, Romain Christiano, Chandramohan Chitraju, Nora Kory, Kenneth Harrison, Joel Haas, Tobias C. Walther, and Robert V. Farese, Jr.
J Lipid Res. Jul 2014; 55(7): 1465–1477. PMCID: PMC4076087.

To obtain a high-certainty lipid droplet proteome, Currie, et al.,1 combined a sophisticated mass spectrometry-based method, called protein correlation profiling (PCP), with sensitive fluorescence microscopy to confirm protein localization to lipid droplets in the yeast Saccharomyces cerevisiae. PCP compares protein abundances in different organelle fractions, as obtained by mass spectrometry, to reduce false positives.

After identifying a set of likely lipid droplet proteins by PCP, the researchers confirmed protein localization to lipid droplets by fluorescence miscroscopy. Using a Hamamatsu ImagEM EM-CCD camera, they were able to co-localize protein labeled with green fluorescent protein in lipid droplets identified by staining with monodansyl pentane.

Of 35 candidate proteins identified by PCP, the researchers localized 30 to lipid droplets by microscopy. Most of those proteins have roles in lipid metabolism. Six of the 30 had never before been identified as lipid droplet proteins. Of those, two were known enzymes with roles in sterol and polyprenol metabolism. The authors went on to demonstrate activity of those two enzymes in lipid droplet fractions. Their paper provides a high-confidence list of proteins that are highly specific to lipid droplets.

THE POSSIBILITIES

Currie, et al., used the high sensitivity of Hamamatsu’s ImagEM EM-CCD camera to localize fluorescently labeled proteins to lipid droplets in S. cerevisiae. Learn how newer sCMOS detectors are making it possible to track intracellular events at video speeds with low-nanometer precision in Exciting Advances Push the Limits of Visualization.

References

  1. Walther and Mann. Mass spectrometry–based proteomics in cell biology. J Cell Biol. Aug 23, 2010; 190(4): 491–500. PMCID: PMC2928005.
  2. Currie, et al. High confidence proteomic analysis of yeast LDs identifies additional droplet proteins and reveals connections to dolichol synthesis and sterol acetylation. J Lipid Res. Jul 2014; 55(7): 1465–1477. PMCID: PMC4076087.
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