Abstract
In both mammals and yeast, intracellular vesicular transport depends on the correct shuttling between membrane and cytosol of the Rab/Ypt small G proteins. Membrane association of these proteins requires prenylation by the Rab geranylgeranyl transferase that recognizes a complex formed by the Rab/Ypt protein and the Rab escort protein (REP). After prenylation the Rab/Ypt protein is delivered to the target membranes by REP. Little is known about the early steps of the Rab-REP complex formation and where this association occurs in the cell. Although prenylation is believed to take place in the cytosol, we show that the yeast Rab escort protein Mrs6 is present in both soluble and particulate fractions of cell extracts. Mrs6p is associated with the heavy microsomal fraction that contains endoplasmic reticulum-Golgi membranes but is absent in the plasma membrane, vacuoles, mitochondria, and microsomal subfraction associated with mitochondria. The solubilization pattern of the particulate pool of Mrs6p implies that this protein is peripherally but tightly associated with membranes via hydrophobic interactions and metal ions. We also report that the C terminus of Mrs6p is important for maintaining the solubility of the protein because its deletion or replacement with the C terminus of RabGDI results in a protein that localizes only to membranes.
Highlights
Rab escort proteins (REP)1 assist in the geranylgeranylation of the Rab/Ypt type of small G proteins, which is catalyzed by Rab geranylgeranyl transferase (GGTase; reviewed in Ref. 1)
A newly synthesized Rab protein is bound by REP, and the complex is recognized by Rab GGTase, which allows a subsequent digeranylgeranylation of a Rab protein to take place
This observation is in agreement with in vitro studies in which a purified mammalian REP-1Rab5 complex was used to study the escorting activity of REP-1 [14]
Summary
Rab escort proteins (REP)1 assist in the geranylgeranylation of the Rab/Ypt type of small G proteins, which is catalyzed by Rab geranylgeranyl transferase (GGTase; reviewed in Ref. 1). Extraction and Proteinase K Treatment of Membrane-bound Mrs6p— Aliquots of total membrane fraction (P100; containing 100 mg of protein) from the wild type strain AR4-6A overexpressing Mrs6p were incubated for 30 min on ice with buffer A (50 mM potassium phosphate, pH 7.6, 10 mM MgCl2, 5 mM dithiothreitol, 1 3 PIM: 0.1 mg/ml chymostatin, 0.2 mg/ml aprotinin, 0.1 mg/ml leupeptin, 2 mg/ml antipain, 0.1 mM benzamidine, and 0.1 mM sodium metabisulfite) either with or without the addition of the reagents described in the text in a total volume of 50 ml.
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