TY  - GEN
UR  - https://archiv.ub.uni-heidelberg.de/volltextserver/10456/
KW  - Src kinase 
KW  -  intracellular transport 
KW  -  acylated proteins 
KW  -  membrane microdomains
TI  - Identification of Components of the Intracellular Transport Machinery of Acylated Proteins by a Genome-wide RNAi Screen
Y1  - 2009///
ID  - heidok10456
AV  - public
A1  - Ritzerfeld, Julia
N2  - Targeting of peripheral membrane proteins to different cellular compartments is often mediated by post-translational fatty acylation. For example, N-terminal SH4-domains containing dual lipid modifications mediate reversible attachment to intracellular membranes of a variety of proteins such as the Src family of kinases. Myristoylation and subsequent palmitoylation of the SH4-domain are not only required for stable membrane anchoring, but are also essential for targeting and transport to the plasma membrane. Their association with membrane microdomains, which are enriched in cholesterol and sphingolipids and moreover contain a specific set of proteins, is crucial for functionality and provides a means of spatio-temporal regulation of acylated proteins. Even though many SH4-domain-containing proteins are functionally quite well characterized, little is known about the intracellular machinery mediating transport to the plasma membrane. It has been hypothesized that their association with membrane microdomains already occurs at the level of intracellular membranes and is a prerequisite for targeting and transport. It was the aim of this study to identify gene products involved in intracellular transport of acylated proteins to the plasma membrane employing a genome-wide RNAi screening approach. For this purpose, we established a stable human model cell line, which simultaneously expresses two distinct plasma-membrane-associated acylated reporter proteins. This cell line was adapted to a high-content screening platform, which is based on reverse transfection of a genome-wide siRNA library and data acquisition by automated widefield microscopy. To analyse imaging data in an unbiased and quantitative manner, an automated single-cell image analysis tool was developed. This tool identifies and compartmentalizes individual cells and determines intensity distributions of the two fluorescent reporter proteins, thus identifying experimental conditions under which intracellular retention of one or both reporter proteins occurs. Primary screening followed by subsequent validation with independent siRNAs resulted in the identification of 60 gene products, which caused intracellular retention of one or both acylated reporter proteins. Interestingly, we were able to identify enzymes involved in lipid homeostasis and microdomain-associated proteins. These findings corroborate the hypothesis that partitioning into membrane microdomains is a crucial step in targeting and transport of acylated proteins to the plasma membrane. Moreover, we were able to identify kinases, phosphatases and other proteins, which may exert regulatory functions in this process. The exact role of these factors in transport of SH4-domain-containing proteins to the plasma membrane will be elucidated in further studies. 
ER  -