Testing Pollen Tube Proteins for In Vivo Binding to Phosphatidic Acid by n-Butanol Treatment and Confocal Microscopy.

The general role of cellular membranes is to provide a barrier and to generate separate reaction spaces. However, additional functions of membrane domains enriched in certain classes of lipids have been discovered, which represent an important area of ongoing research. Such membrane domains can be found in cells at different size scales (e.g., nanodomains, microdomains), represent membrane regions with special physical properties and play important roles in the direct or indirect propagation of signaling processes. Domain formation within the plasma membrane (PM) does not only involve the accumulation of specific lipids, but also the recruitment of specific transmembrane or PM-associated peripheral proteins. Phosphatidic acid (PA) is increasingly recognized as an important signaling lipid and component of PM domains. This lipid is involved in the regulation not only of biotic or abiotic stress responses, but also of pollen tube tip growth and of other forms of polar cell expansion. Although many PA-binding proteins have been characterized, a conserved PA interaction motif could not be identified in these proteins. Consequently, protein binding to PA cannot be predicted based on sequence analysis, but has to be biochemically tested using lipid strip or liposome assays. Although these assays are often informative, they are generally based on the use of artificial model membranes, which compared to natural membranes contain fewer lipid types often at non-physiological concentrations. In this chapter, we describe an alternative in vivo assay that can be employed to analyze protein binding to PA at the PM of normally elongating tobacco pollen tubes. This assay is based on the use of n-butanol (n-ButOH), which inhibits phospholipase D (PLD) and thereby blocks a major biosynthetic pathway that generates PA within the PM from substrates like phosphatidylcholine (PC) or phosphatidylethanolamine (PE). PLD inhibition reduces the PA content of the PM and consequently the level of PM association of PA-binding proteins, which can be analyzed using fluorescence microscopy. Methods enabling n-ButOH treatment of cultured tobacco pollen tubes expressing YFP-tagged PA-binding proteins as well as the quantitative determination of the PM association of these proteins are described.