The sorting determinant guiding Hsp150 to the COPI-independent transport pathway in yeast

The COPI coatomer is thought to be required in yeast directly for retrograd e transport from the Golgi to the endoplasmic reticulum (ER), and directly or indirectly for ER-to-Golgi transport. Unexpectedly, the secretory glycop roteins Hsp150 and invertase have been found not to require COPI for ER exi t. The features according to which cargo proteins are selected for the COPI -independent pathway are not known. The ER form of Hsp150 has three distinc t domains: an N-terminal fragment of 54 amino acids (subunit I) is followed by 11 repeats of a 19 amino acid peptide plus a unique C-terminal fragment of 114 amino acids (subunit II). By fusing heterologous proteins to differ ent Hsp150 domains and expressing them in sec21-1 and sec21-3 mutants with temperature-sensitive mutations in the gamma-COPI subunit, we show here tha t the repeats of subunit II function as sorting determinants for COPI-indep endent ER exit. The C-terminal fragment of Hsp150 could be replaced by E. c oli beta-lactamase or rat nerve growth factor receptor ectodomain (NGFRe), and subunit I could be deleted, without inhibiting COPI-independent transpo rt. However, when the repetitive region was omitted and beta-lactamase was fused directly to the C terminus of subunit I, COPI was required for effici ent ER exit. Mass spectroscopic analysis demonstrated that both subunit I a nd II of Hsp150 were extensively O-glycosylated, suggesting that the O-glyc osylation pattern was not decisive for cargo selection.