title: Sorting of a HaloTag protein that has only a signal peptide sequence into exocrine secretory granules without protein aggregation.
abstract: The mechanism involved in the sorting and accumulation of secretory cargo proteins, such as amylase, into secretory granules of exocrine cells remains to be solved. To clarify that sorting mechanism, we expressed a reporter protein HaloTag fused with partial sequences of salivary amylase protein in primary cultured parotid acinar cells. We found that a HaloTag protein fused with only the signal peptide sequence (Met(1)-Ala(25)) of amylase, termed SS25H, colocalized well with endogenous amylase, which was confirmed by immunofluorescence microscopy. Percoll-density gradient centrifugation of secretory granule fractions shows that the distributions of amylase and SS25H were similar. These results suggest that SS25H is transported to secretory granules and is not discriminated from endogenous amylase by the machinery that functions to remove proteins other than granule cargo from immature granules. Another reporter protein, DsRed2, that has the same signal peptide sequence also colocalized with amylase, suggesting that the sorting to secretory granules is not dependent on a characteristic of the HaloTag protein. Whereas Blue Native PAGE demonstrates that endogenous amylase forms a high-molecular-weight complex, SS25H does not participate in the complex and does not form self-aggregates. Nevertheless, SS25H was released from cells by the addition of a β-adrenergic agonist, isoproterenol, which also induces amylase secretion. These results indicate that addition of the signal peptide sequence, which is necessary for the translocation in the endoplasmic reticulum, is sufficient for the transportation and storage of cargo proteins in secretory granules of exocrine cells.
authors.affiliation: Dept. of Physiology, Nihon Univ., School of Dentistry at Matsudo, 2-870-1 Sakaecho-nishi, Matsudo, Chiba, 271-8587, Japan. yoshigaki.junko@nihon-u.ac.jp.
authors.forename: Junko
authors.initials: J
authors.lastname: Fujita-Yoshigaki
authors.forename: Miwako
authors.initials: M
authors.lastname: Matsuki-Fukushima
authors.forename: Megumi
authors.initials: M
authors.lastname: Yokoyama
authors.forename: Osamu
authors.initials: O
authors.lastname: Katsumata-Kato
mesh_terms: D061354:Acinar Cells / Q000166:cytology / Q000502:physiology*; D000818:Animals; D002478:Cells, Cultured; D015180:Electrophoresis, Gel, Two-Dimensional; D005786:Gene Expression Regulation; D008297:Male; D010179:Pancreas / Q000166:cytology*; D010306:Parotid Gland / Q000166:cytology* / Q000378:metabolism; D021382:Protein Sorting Signals / Q000502:physiology*; D021381:Protein Transport; D051381:Rats; D017207:Rats, Sprague-Dawley; D022142:Secretory Vesicles / Q000378:metabolism*; D013194:Staining and Labeling
chemical_list: D021382:Protein Sorting Signals
keywords: exocrine glands; exocytosis; regulated secretion; secretory granules; sorting mechanism