The internalization of yeast Ste6p follows an ordered series of events involving phosphorylation, ubiquitination, recognition and endocytosis.
A general pathway for the internalization of plasma membrane proteins that involves phosphorylation, ubiquitination, recognition and endocytosis has recently emerged from multiple studies in yeast. We refer to this series of events as the PURE pathway. Here we investigate whether the yeast a-factor transporter Ste6p, an ATP-binding cassette protein, utilizes the PURE pathway. Deletion of a 52-amino acid sequence (the 'A box') within the linker region of Ste6p has previously been shown to block ubiquitination and endocytosis (Kolling R, Losko S. EMBO J 1997; 16:2251-2261). Using wild-type and mutant forms of GFP-tagged Ste6p, we identified two residues (T(613) and S(623)) within the A box as likely sites of Ste6p phosphorylation important for internalization. Mutation of these residues to alanine blocked ubiquitination and endocytosis of Ste6p, similar to the effect of deleting the entire A box, while substitution with glutamic acid (to mimic phosphorylation) suppressed the ubiquitination and endocytic defects. Importantly, a translational fusion of monoubiquitin to the C-terminus of Ste6p-T(613)A, S(623)A or Ste6p-DeltaA restored endocytosis, providing strong evidence that the role of phosphorylation is to direct ubiquitination, which in turn is a critical signal for Ste6p internalization. We also identified multiple (five) lysine residues in the linker that are important for Ste6p ubiquitination. Our results demonstrate that Ste6p follows the PURE pathway and that GFP-tagged Ste6p provides a powerful model protein for studies of endocytosis and post-endocytic events in yeast.
Kelm
Kellie B
KB
Department of Cell Biology, The Johns Hopkins University School of Medicine, 725 N Wolfe St, Baltimore, MD 21205, USA.
Huyer
Gregory
G
Huang
Joseph C
JC
Michaelis
Susan
S
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D004705:Endocytosis; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation; D021381:Protein Transport; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014452:Ubiquitins / Q000378:metabolism*; D015003:Yeasts / Q000737:chemistry / Q000166:cytology* / Q000235:genetics / Q000378:metabolism*
D018528:ATP-Binding Cassette Transporters; D000596:Amino Acids; D006023:Glycoproteins; D008565:Membrane Proteins; C489277:STE6 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014452:Ubiquitins