The Yeast Vps10p Cytoplasmic Tail Mediates Lysosomal Sorting in Mammalian Cells and Interacts with Human GGAs

André Dennes,Peder Madsen,Morten S Nielsen,Claus M Petersen,Regina Pohlmann

doi:10.1074/jbc.m112295200

André Dennes, Peder Madsen + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m112295200

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Abstract

Yeast Vps10p is a receptor for transport of the soluble vacuolar hydrolase carboxypeptidase Y to the lysosome-like vacuole. Its functional equivalents in mammalian cells are the mannose 6-phosphate receptors that mediate sorting to lysosomes of mannose 6-phosphate-containing lysosomal proteins. A chimeric receptor was constructed by substituting the cytoplasmic domain of M(r) 300,000 mannose 6-phosphate receptor with the Vps10p cytoplasmic tail. Expression of the chimera in cells lacking endogenous mannose 6-phosphate receptors resulted in a subcellular receptor distribution and an efficiency in sorting of lysosomal enzymes similar to that of the wild type M(r) 300,000 mannose 6-phosphate receptor. Moreover, the cytoplasmic tail of the Vps10p was found to interact with GGA1 and GGA2, two mammalian members of a recently discovered family of clathrin-binding cytosolic proteins that participate in trans-Golgi network-endosome trafficking in both mammals and yeast. Our findings suggest a conserved machinery for Golgi-endosome/vacuole sorting and may serve as a model for future studies of yeast proteins.

Highlights

Yeast Vps10p is a receptor for transport of the soluble vacuolar hydrolase carboxypeptidase Y to the lysosomelike vacuole
The receptors were expressed in mouse embryonic fibroblasts that lack endogenous MPR46 and MPR300 [42] and missort 98% of soluble lysosomal enzymes to the medium
Transfected clones were selected, and the level of the chimera expression was quantified in permeabilized cells by measuring the binding of a monoclonal antibody directed against the lumenal domain of MPR300

Summary

EXPERIMENTAL PROCEDURES

Receptor Constructs—The chimera was named corresponding to the origin of the lumenal, transmembrane, and cytoplasmic domain of either the MPR300 (L) or the Vps10p (V) (compare Fig. 1). Expression Levels—For determination of the relative amounts of wild type MPR300 and MPR300-Vps10p chimera, the monoclonal mouse antibody 2C2 recognizing the luminal epitope of human MPR300 was used [38]. The day the plates where incubated with 5 ␮g of 2C2 antibody/ml of binding medium (Dulbecco’s minimal essential medium, 7.5% heat-inactivated fetal calf serum, and 20 mM Hepes/KOH, pH 7.4) for 2 h at 4 °C in the presence of 0.5% saponin. Biotin-labeled sheep anti-mouse antibody (Amersham Biosciences, Inc.) in blocking buffer (1: 300) was allowed to bind for 1 h. For the human MPR300 lumenal domain the mouse monoclonal antibody 2C2 was used, and the human MPR46 was detected using a specific serum from goat. The proteins precipitated from cells were dissociated from the Sepharose beads by the addition of surplus glutathione (Sigma) and subsequently subjected to Western blotting detecting MPR300. The amounts of MPR300 protein found in lysates prior to precipitation and the quantity isolated by affinity beads were compared by SDS-PAGE

RESULTS

Cell line

DISCUSSION