Abstract
A number of inherited lysosomal diseases are known to result from missorting of lysosomal proteins. Considerable attention has been directed toward an understanding of this sorting pathway, and it has become apparent that different mechanisms are used for the sorting of lysosomal membrane and soluble proteins. Protein sorting to the yeast vacuole/lysosome provides a simple model system to study this process. We have mapped the first sorting signal in a vacuolar membrane protein, repressible alkaline phosphatase, and have shown it to be both necessary and sufficient for vacuolar delivery of this enzyme. The sorting information is confined to the transmembrane and cytoplasmic tail region of this type II integral membrane protein. The location of this sorting signal provides an explanation for some of the differences observed between membrane and soluble vacuolar protein sorting.
Highlights
Protein sorting to the yeast vacuolejlysosome provides a simple model system to study this process
The secretory pathway [4] has long been used as a model system to study the processes involved in protein sorting, and much of this work has focused on the delivery of proteins to the mammalian lysosome [5]
An analysis of protein sorting to the yeast vacuole, which is analogous to the mammalian lysosome, has been useful in identifying both the c&acting signals involved in targeting proteins to the vacuole and the trans-acting components of the machinery that mediate the sorting process (7-l 1)
Summary
Materials-Lyticase was obtained from Enzogenetics (Corvallis, OR), Tran?‘S-label was from ICN Radiochemicals (Irvine, CA), o[*-. The ALP-Inv191 fusion utilized a BglII site in PHOS and was cloned as an EcoRI-BglII fragment into the EcoRI-BamHI sites of plasmid pSEYC306 [7]. The presence of the BamHI site resulted in the introduction of an additional amino acid residue (aspartic acid) at position 21 of the hybrid protein. 15 min before the addition of Tran&-label. Invertase antiserum was used to precipitate the hybrid proteins as described previously [9]. DKY6280 harboring plasmid pDKY1 was converted to spheroplasts and labeled as described previously [10]. Chased, and separated into intracellular (spheroplast) and extracellular (periplasm and medium) fractions and immunoprecipitated twice with antiserum to ALP. Golgi-specific carbohydrate modifications, the 30-min chase sample was divided in half after the first precipitation with ALP antiserum
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