Elsevier

Analytical Biochemistry

Volume 347, Issue 2, 15 December 2005, Pages 165-175
Analytical Biochemistry

Review
Phosphatidylserine decarboxylases as genetic and biochemical tools for studying phospholipid traffic

https://doi.org/10.1016/j.ab.2005.03.017Get rights and content

Section snippets

Assay systems for measurement of PtdSer decarboxylase activity

Three basic assay systems are used to measure the catalytic activity of PtdSer decarboxylases as summarized in Fig. 1. One of the simplest assay systems is based upon that originally described by Kanfer and Kennedy [7] for measuring the enzyme activity in extracts of Escherichia coli. The assay for Psd1p from yeast and other eukaryotes uses a Ptd[1′-14C]Ser substrate that is added to an enzyme preparation in a suitable tube with a gas-tight stopper [1]. Specialized rubber stoppers that enable

Overview of the selective use of PtdSer decarboxylase mutants

The cloning of PSD genes in yeast not only provided important information about the primary structure and organization of the genes and the encoded proteins but also created important opportunities for genetic manipulation of strains to isolate interesting new mutants in lipid metabolism and reveal new information about lipid transport [6]. Fig. 2 outlines some of the major features of PtdSer and PtdEtn traffic in yeast and mammalian cells. The pathways for lipid transport have been

PtdSer transport to Psd1p: from intact cells to reconstitution with isolated organelles

The decarboxylation catalyzed by Psd1p at the inner mitochondrial membrane has proven to be extremely useful as an indicator reaction for measuring PtdSer transport to this organelle [28]. The products of the reaction, either PtdEtn recovered in lipid extracts or CO2 trapped on filter paper, can be easily quantified by the procedures described earlier. While the PtdEtn formation from radiolabeled Ser or PtdSer can be used in transport studies ranging from whole cells to isolated organelles, the

Intact cells

The decarboxylation of nascent PtdSer by Psd2p can be exploited to study PtdSer movement from its site of synthesis to the Golgi in yeast cells [6]. Using strains with a psd1Δ mutation ensures that nearly all of the PtdEtn formation from radiolabeled Ser occurs via the action of Psd2p. The lipid transport process is measured by culturing the cells in minimal medium supplemented with 10 μCi/ml [3H]Ser for varying periods of time, followed by lipid extraction and analysis using thin-layer

Concluding comments

The PtdSer decarboxylases have emerged as important biochemical and genetic tools for the study of interorganelle phospholipid traffic. The application of the approaches described in this review has thus far yielded important information about six components involved in PtdSer transport and a growing body of biochemical detail about the processes. These advances should be viewed as only modest beginnings toward the goal of unraveling structural and mechanistic descriptions of the lipid aspect

Acknowledgment

This work was supported by the National Institutes of Health Grant 2R37 GM 32453.

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