Sorting of a HaloTag protein that has only a signal peptide sequence into exocrine secretory granules without protein aggregation

Abstract

Fujita-Yoshigaki J, Matsuki-Fukushima M, Yokoyama M, Katsumata-Kato O. Sorting of a HaloTag protein that has only a signal peptide sequence into exocrine secretory granules without protein aggregation. Am J Physiol Gastrointest Liver Physiol 305: G685-G696, 2013. First published September 12, 2013 doi:10.1152/ajpgi.00093.2013The 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-molecularweight 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 ␤-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.

<p>Current insights of inspiratory muscle training on the cardiovascular system: a systematic review with meta-analysis</p>

“…The initial search led to the identification of 975 studies, among which 855 were considered potentially relevant based on the title and abstract. Twenty-nine potentially relevant studies were retrieved for detailed analysis 8,11,18–34. Only 10 articles met the eligibility criteria 9,35–43…”

HaloTag Technology: A Versatile Platform for Biomedical Applications

“…Using similar methods, the mechanism of accumulation into secretory granules of exocrine cells by amylase was visualized using a partial sequence of salivary amylase protein fused with HaloTag (Figure 5 ). 92 In this study, two amylase-modified HaloTag proteins were investigated after ensuring that the HaloTag conjugation was successful (Figure 5 A,B). Through characterization using fluorescent microscopy, they showed that the HaloTag–amylase protein was similar to endogenous amylase, as it colocalized in similar cellular regions (Figure 5 C).…”

“…Scale bar = 10 μm. Reprinted with permission from ref ( 92 ). Copyright 2013 the American Physiological Society.…”

Involvement of myristoylated alanine-rich C kinase substrate phosphorylation and translocation in cholecystokinin-induced amylase release in rat pancreatic acini

“…Preparation of the pancreatic acini. The pancreatic acini were prepared as described previously (17,35,62) with some modifications. Sprague-Dawley rats (males, 200 -250 g) were anesthetized intraperitoneally with pentobarbital (50 mg/kg), and the pancreas was removed and placed in a small volume of Krebs-Ringer bicarbonate (KRB) solution with the following composition: 116 mM NaCl, 5.4 mM KCl, 0.8 mM MgSO 4, 1.8 mM CaCl2, 0.96 mM NaH2PO4, 25 mM NaHCO 3, 5 mM HEPES (pH 7.4), and 11.1 mM glucose.…”