Use of Biotin-Labeled Geranyl Pyrophosphate for Analysis of Ykt6 Geranylgeranylation.

Functionally derivatized analogs of prenyl lipids are valuable tools for the detection and analysis of prenylated proteins. Using a biotinylated analog of geranylgeranyl, we previously identified Ykt6 as a substrate for a novel protein prenyltransferase, termed geranylgeranyltransferase type III (GGTase-III). Ykt6 is an evolutionarily highly conserved SNARE protein that regulates multiple intracellular trafficking pathways, including intra-Golgi trafficking and autophagosome-lysosome fusion. Unlike most SNARE proteins, which are membrane-anchored via a C-terminal transmembrane region, Ykt6 is uniquely attached to the membrane by lipid modifications at two conserved cysteines near the C-terminus (Cys194 and Cys195 in human Ykt6). Cys195, the fourth residue from the C-terminus, is farnesylated by farnesyltransferase (FTase), while Cys194 is geranylgeranylated by GGTase-III. These two enzymes sequentially transfer farnesyl and geranylgeranyl groups to Ykt6, producing a doubly prenylated form of Ykt6. Double prenylation is essential for the function of Ykt6 and is crucial for the maintenance of the Golgi apparatus and autophagosome clearance. Here we describe the use of the biotin-labeled geranylgeranyl analog to identify Ykt6 as a GGTase-III substrate.