Difference in distribution of membrane proteins between low- and high-density secretory granules in parotid acinar cells

Fujita-Yoshigaki, Junko; Katsumata, Osamu; Matsuki, Miwako; Yoshigaki, Tomoyoshi; Furuyama, Shunsuke; Saito, Hiroshi

doi:10.1016/j.bbrc.2006.03.130

Cited by 17 publications

(13 citation statements)

References 25 publications

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“…The density of SGs is thought to become higher after generation from the TGN, which is called the maturation process (49). During maturation, the components of the cargo and membrane proteins change (16,22,49). Some lysosomal proteins have been reported to be removed from immature granules after generation (24).…”

Section: Resultsmentioning

confidence: 99%

“…The SGs of rat parotid acinar cells were isolated as previously described (16,22). At 2 days after infection with the adenovirus for expression of SS25H, cultured parotid acinar cells were harvested with buffer A (300 mM sucrose, 1 mM MgCl 2, 1 mM dithiothreitol, 1ϫ Complete EDTA-free, 20 mM MOPS/NaOH, pH 7.0).…”

Section: Quantitative Colocalization Analysis 3 (Colocalization Parammentioning

confidence: 99%

“…The removed proteins are secreted immediately (constitutive-like secretory pathway) or in response to low-level stimulation (minor-regulated pathway) (2,10,49) or are transported to other subcellular compartments, such as lysosomes and endosomes (7,24,26). Through the selective removal of nongranular proteins, immature granules are converted into mature granules (2,16,22,24,46).…”

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confidence: 99%

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

Fujita-Yoshigaki

Matsuki-Fukushima

Yokoyama³

et al. 2013

American Journal of Physiology-Gastrointestinal and Liver Physi

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Quantitative Colocalization Analysis 3 (Colocalization Parammentioning

confidence: 99%

mentioning

confidence: 99%

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

Fujita-Yoshigaki

Matsuki-Fukushima

Yokoyama³

et al. 2013

American Journal of Physiology-Gastrointestinal and Liver Physi

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“…To study the mechanism of the generation and maturation of SGs in parotid acinar cells, we previously examined the presence of membrane proteins in the SG fractions prepared by Percolldensity gradient centrifugation. We found that the distribution of membrane proteins among the fractions is dramatically different (2). Syntaxin 6 and vesicle associated membrane protein 4(VAMP4)were abundant in immature granule fractions while they were not detected in mature granule fractions.…”

Section: Introductionmentioning

confidence: 91%

<b>Syntaxin 6 is Involved in the Maintenance of Secretory Granules in Parotid Acinar Cells </b>

2017

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Secretory granules (SGs) of salivary glands are considered to be generated as an immature granule and to mature by condensing their contents. Syntaxin 6 and vesicle associated membrane protein 4 (VAMP4) are detected in the membrane fractions of immature but not mature SGs, suggesting that syntaxin 6 and VAMP4 are transported from SGs to other organelles during the process of granule maturation. To study the role of syntaxin 6 in the biogenesis of granules, we transfected syntaxin 6 fused with enhanced green fluorescent protein(EGFP)into primary cultured parotid acinar cells. The EGFPsyntaxin 6 signal overlapped with that of an early endosome marker, early endosome antigen 1 (EEA1), which was detected using immunofluorescence. The localization of EGFP-fused VAMP4 was also similar to that of EEA1, suggesting that syntaxin 6 and VAMP4 are transported from immature SGs to early endosomes. Transfection of a syntaxin 6 mutant that lacks a SNARE-binding domain caused a decrease in the number of SGs although the localization of the mutant was unchanged from that of the wild type syntaxin 6. These results suggest that syntaxin 6 has an important role in the maintenance of SGs in parotid acinar cells.

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“…Importantly, populations of vesicles are distinguished by the presence of specific combinations of coat proteins, such as clathrin, Adaptor Proteins (AP1-4), FAPP1/2, GGAs, ARF, v-snares, and synaptotagmin. In the parotid gland, VAMP2, VAMP8, syntaxin4/6, and synaptotagmin decorate the cytoplasmic side of secretory granules (Fujita-Yoshigaki et al 2006;Wang et al 2007). These different coat proteins on different vesicles direct the vesicles to the correct target membranes.…”

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Phosphatidylinositol Bisphosphate Mediated Sorting of Secretory Granule Cargo

Darling¹,

Venkatesh²,

Goyal³

et al. 2012

Crosstalk and Integration of Membrane Trafficking Pathways

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Difference in distribution of membrane proteins between low- and high-density secretory granules in parotid acinar cells

Cited by 17 publications

References 25 publications

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

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

<b>Syntaxin 6 is Involved in the Maintenance of Secretory Granules in Parotid Acinar Cells </b>

Phosphatidylinositol Bisphosphate Mediated Sorting of Secretory Granule Cargo

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