The sequence NPFXD defines a new class of endocytosis signal in Saccharomyces cerevisiae.

Abstract

Abstract. The yeast membrane protein Kex2p uses a tyrosine-containing motif within the cytoplasmic domain for localization to a late Golgi compartment. Because Golgi membrane proteins mislocalized to the plasma membrane in yeast can undergo endocytosis, we examined whether the Golgi localization sequence or other sequences in the Kex2p cytoplasmic domain mediate endocytosis. To assess endocytic function, the Kex2p cytoplasmic domain was fused to an endocytosis-defective form of the a-factor receptor, Ste2p. Like intact Ste2p, the chimeric protein, Stex22p, undergoes rapid endocytosis that is dependent on clathrin and End3p. Uptake of Stex22p does not require the Kex2p Golgi localization motif. Instead, the sequence NPFSD, located 37 amino acids from the COOH terminus, is essential for Stex22p endocytosis. Internalization was abolished when the N, P, or F residues were converted to alanine and severely impaired upon conversion of D to A. NPFSD restored uptake when added to the COOH terminus of an endocytosis-defective Ste2p chimera lacking lysine-based endocytosis signals present in wild-type Ste2p. An NPF sequence is present in the cytoplasmic domain of the a-factor receptor, Ste3p. Mutation of this sequence prevented pheromone-stimulated endocytosis of a truncated form of Ste3p. Our results identify NPFSD as a clathrin-dependent endocytosis signal that is distinct from the aromatic amino acid-containing Golgi localization motif and lysinebased, ubiquitin-dependent endocytosis signals in yeast. C LATHRIN-mediated endocytosis of plasma membranereceptors promotes the rapid and efficient uptake of receptor-bound ligands, typically nutrients and signaling molecules important for cell growth and differentiation. Plasma membrane proteins subject to efficient endocytosis contain specific, cytoplasmically disposed amino acid sequences that are necessary for uptake (for review see Trowbridge et al., 1993). Such endocytic targeting signals often contain an aromatic amino acid (tyrosine or phenylalanine) and serve to direct proteins into clathrincoated pits. The critical importance of aromatic amino acids in the targeting sequences has been established by mutational studies (for review see Trowbridge et al., 1993). For example, mutation of tyrosine 807 in the low-density lipoprotein (LDL) 1 receptor disrupts clathrin-coated pit localization and thereby prevents uptake from the plasma membrane (Davis et al., 1986(Davis et al., , 1987 the influenza hemagglutinin protein dramatically increases the efficiency of internalization via clathfin-coated pits (Lazarovits and Roth, 1988). In addition to the aromatic amino acid--containing motifs, there are also endocytic targeting signals that lack aromatic amino acids but appear to mediate internalization through clathrin-coated pits. Examples of these signals include di-leucine motifs in the CD3 subunits of the T cell antigen receptor (Letourneur and Klausner, 1992) and a lysine-containing signal in the yeast a-factor receptor, Ste2p (Rohrer et al., 1993).The location of endocytic t...

The Arf activator Gea2p and the P-type ATPase Drs2p interact at the Golgi in Saccharomyces cerevisiae

“…5B, ∆1283-1355). This result indicates that the two NPFXD motifs, potential binding sites for the Pan1/End3/Sla1 complex (Howard et al, 2002;Tan et al, 1996), and half of the highly conserved domain are not required for full function of Drs2p. However, the additional removal of the GFAFSQAEE sequence (∆1274-1355) strongly abrogated complementation.…”

The Yeast Actin Cytoskeleton: from Cellular Function to Biochemical Mechanism

“…The early recruitment process may begin with cytosolic regions of membrane receptors, possibly after being modified by kinases and ubiquitin ligases, associating with endocytic machinery (147,156,323,346,362). Alternatively, some of the early endocytic machinery may reside in patches at the cell cortex and recruit and concentrate receptors.…”

Eps15 Is Recruited to the Plasma Membrane upon Epidermal Growth Factor Receptor Activation and Localizes to Components of the Endocytic Pathway during Receptor Internalization

“…A number of observations have recently linked eps15, eps15R, and other EH-containing proteins to coated pits-mediated internalization. 1) eps15 (Tebar et al, 1996;Van Delft et al, 1997b) and eps15R (Coda et al, 1998) colocalize with markers of the plasma membrane clathrin-coated pits and vesicles; 2) by electron microscopy, eps15 is found at the rim of budding coated vesicles (Tebar et al, 1996); 3) eps15 and eps15R are constitutively associated with the clathrin adaptor protein complex AP-2 (Benmerah et al, 1995(Benmerah et al, , 1996Iannolo et al, 1997;Van Delft et al, 1997b;Coda et al, 1998); 4) a putative 160-kDa EH-containing protein is associated with the ␥-subunit of the Golgi adaptor complex AP-1 (Robinsons and Page, 1996); 5) End3p, an EH-containing yeast protein, is essential for endocytosis of the ␣-factor receptor (Benedetti et al, 1994); 6) mutations in the EH domains of another yeast protein, Pan1p, impair endocytosis (Wendland et al, 1996;Tang et al, 1997); in addition, Pan1p functions as an adaptor protein involved in the assembly of protein: protein interactions essential for endocytosis (Wendland and Emr, 1998); 7) the amino acid motif NPF (Asn-ProPhe) is a ligand for the EH domain and functions as an internalization motif in yeast (Tan et al, 1996); 8) microinjection of anti-eps15 or anti-eps15R antibodies inhibits endocytosis of EGF and TfR (Carbone et al, 1997, Benmerah et al, 1998; and 9) overexpression of a dominant negative mutant of eps15, comprising only its AP-2 binding region, is also able to inhibit EGFR internalization (Carbone et al, 1997).…”