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 retrograde transport from the Golgi to the endoplasmic reticulum (ER), and directly or indirectly for ER-to-Golgi transport. Unexpectedly, the secretory glycoproteins Hsp150 and invertase have been found not to require COPI for ER exit. The features according to which cargo proteins are selected for the COPI-independent pathway are not known. The ER form of Hsp150 has three distinct 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 different Hsp150 domains and expressing them in sec21-1 and sec21-3 mutants with temperature-sensitive mutations in the gamma-COPI subunit, we show here that the repeats of subunit II function as sorting determinants for COPI-independent ER exit. The C-terminal fragment of Hsp150 could be replaced by E. coli beta-lactamase or rat nerve growth factor receptor ectodomain (NGFRe), and subunit I could be deleted, without inhibiting COPI-independent transport. However, when the repetitive region was omitted and beta-lactamase was fused directly to the C terminus of subunit I, COPI was required for efficient ER exit. Mass spectroscopic analysis demonstrated that both subunit I and II of Hsp150 were extensively O-glycosylated, suggesting that the O-glycosylation pattern was not decisive for cargo selection.
Suntio
T
T
Institute of Biotechnology, PO Box 58, Finland.
Shmelev
A
A
Lund
M
M
Makarow
M
M
D000595:Amino Acid Sequence; D001692:Biological Transport; D020755:Coat Protein Complex I / Q000502:physiology*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics; D006023:Glycoproteins; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013058:Mass Spectrometry; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D020800:Receptor, Nerve Growth Factor / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism
D020755:Coat Protein Complex I; D005656:Fungal Proteins; D006023:Glycoproteins; C074229:HSP150 protein, S cerevisiae; D006360:Heat-Shock Proteins; D020800:Receptor, Nerve Growth Factor; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D001618:beta-Lactamases