Evidence for colocalization and interaction between 37 and 39 kDa isoforms of secretory carrier membrane proteins (SCAMPs)

1997 ◽  
Vol 110 (13) ◽  
pp. 1533-1541 ◽  
Author(s):  
T.T. Wu ◽  
J.D. Castle
Keyword(s):  
Membrane Proteins ◽  
Acinar Cells ◽  
Chemical Crosslinking ◽  
Vesicle Membrane ◽  
Parotid Acinar Cells ◽  
Single Protein ◽  
Transport Vesicle ◽  
Potential Formation ◽  
Secretory Carrier Membrane Proteins

Secretory carrier membrane proteins (SCAMPs) are proteins of post-Golgi recycling carriers, including regulated secretory organelles. The two major size variants, SCAMP1 (37 kDa) and SCAMP2 (39 kDa), extensively colocalize in membranes of fibroblasts and parotid acinar cells based on immunocytochemistry and velocity centrifugation, although the relative amounts of each variant may differ in selected organelles. SCAMP1, and to a lesser extent, SCAMP2, are substrates for chemical crosslinking in situ, and the recognizable crosslinking products of SCAMP1 suggest potential formation of homomultimers. SCAMP1 and SCAMP2 can be co-immunoprecipitated following detergent solubilization, using antibodies that specifically react with only one of the variants. Both the localization and interactions of SCAMPs are reiterated using transfected SCAMP1 that is epitope tagged (myc) at either the NH2 or COOH terminus and an anti-myc antibody. Like other transport vesicle membrane proteins, SCAMPs form complexes that apparently include homomultimers. Furthermore, these studies suggest that both SCAMP1 and SCAMP2 may function together in a single protein complex.

scholarly journals Tyrosine Phosphorylation of Selected Secretory Carrier Membrane Proteins, SCAMP1 and SCAMP3, and Association with the EGF Receptor

1998 ◽  
Vol 9 (7) ◽  
pp. 1661-1674 ◽  
Author(s):  
Theodore T. Wu ◽  
J. David Castle
Keyword(s):  
Membrane Proteins ◽  
Egf Receptor ◽  
Tyrosine Phosphatase ◽  
Chinese Hamster ◽  
Tyrosine Residue ◽  
Cell Interior ◽  
Murine Fibroblasts ◽  
Secretory Carrier Membrane Proteins

Secretory carrier membrane proteins (SCAMPs) are ubiquitously expressed proteins of post-Golgi vesicles. In the presence of the tyrosine phosphatase inhibitor vanadate, or after overexpression in Chinese hamster ovary (CHO) cells, SCAMP1 and SCAMP3 are phosphorylated selectively on tyrosine residue(s). Phosphorylation is reversible after vanadate washout in situ or when isolated SCAMP3 is incubated with the recombinant tyrosine phosphatase PTP1B. Vanadate also causes the partial accumulation of SCAMP3, but not SCAMP1, in “patches” at or near the cell surface. A search for SCAMP kinase activities has shown that SCAMPs 1 and 3, but not SCAMP2, are tyrosine phosphorylated in EGF-stimulated murine fibroblasts overexpressing the EGF receptor (EGFR). EGF catalyzes the progressive phosphorylation of the SCAMPs up to 1 h poststimulation and may enhance colocalization of the EGFR and SCAMP3 within the cell interior. EGF also induces SCAMP–EGFR association, as detected by coimmunoprecipitation, and phosphorylation of SCAMP3 is stimulated by the EGFR in vitro. These results suggest that phosphorylation of SCAMPs, either directly or indirectly, may be functionally linked to the internalization/down-regulation of the EGFR.

scholarly journals Zymogen granules of mouse parotid acinar cells are acidified in situ in an ATP-dependent manner

1988 ◽  
Vol 253 (1) ◽  
pp. 267-269 ◽  
Author(s):  
Robert C. De Lisle ◽  
Robin Steinberg ◽  
John A. Williams
Keyword(s):  
Acinar Cells ◽  
Dependent Manner ◽  
Zymogen Granules ◽  
Parotid Acinar Cells

Molecular identification of Ca2+-activated K+ channels in parotid acinar cells

2003 ◽  
Vol 284 (2) ◽  
pp. C535-C546 ◽  
Author(s):  
Keith Nehrke ◽  
Claire C. Quinn ◽  
Ted Begenisich
Keyword(s):  
Single Channel ◽  
Acinar Cells ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Parotid Acinar Cells ◽  
Large Conductance Channel ◽  
Intracellular Ca

We used molecular biological and patch-clamp techniques to identify the Ca2+-activated K+ channel genes in mouse parotid acinar cells. Two types of K+ channels were activated by intracellular Ca2+ with single-channel conductance values of 22 and 140 pS (in 135 mM external K+), consistent with the intermediate and maxi-K classes of Ca2+-activated K+ channels, typified by the mIK1 ( Kcnn4) and mSlo ( Kcnma1) genes, respectively. The presence of mIK1 mRNA was established in acinar cells by in situ hybridization. The electrophysiological and pharmacological properties of heterologously expressed mIK1 channels matched those of the native current; thus the native, smaller conductance channel is likely derived from the mIK1 gene. We found that parotid acinar cells express a single, uncommon splice variant of the mSlo gene and that heterologously expressed channels of this Slo variant had a single-channel conductance indistinguishable from that of the native, large-conductance channel. However, the sensitivity of this expressed Slo variant to the scorpion toxin iberiotoxin was considerably different from that of the native current. RT-PCR analysis revealed the presence of two mSlo β-subunits ( Kcnmb1 and Kcnmb4) in parotid tissue. Comparison of the iberiotoxin sensitivity of the native current with that of parotid mSlo expressed with each β-subunit in isolation and measurements of the iberiotoxin sensitivity of currents in cells from β1 knockout mice suggest that parotid acinar cells contain approximately equal numbers of homotetrameric channel proteins from the parotid variant of the Slo gene and heteromeric proteins composed of the parotid Slo variant in combination with the β4-subunit.

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

2006 ◽  
Vol 344 (1) ◽  
pp. 283-292 ◽  
Author(s):  
Junko Fujita-Yoshigaki ◽  
Osamu Katsumata ◽  
Miwako Matsuki ◽  
Tomoyoshi Yoshigaki ◽  
Shunsuke Furuyama ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Secretory Granules ◽  
Acinar Cells ◽  
High Density ◽  
Parotid Acinar Cells

Use of a crystallization robot to set up sitting-drop vapor-diffusion crystallization andin situcrystallization screens

2000 ◽  
Vol 33 (2) ◽  
pp. 344-349 ◽  
Author(s):  
Christopher F. Snook ◽  
Michael D. Purdy ◽  
Michael C. Wiener
Keyword(s):  
Membrane Proteins ◽  
Protein Crystallization ◽  
Integral Membrane Proteins ◽  
Vapor Diffusion ◽  
Liquid Handling ◽  
General Utility ◽  
Crystallization Experiments ◽  
Automated Screening ◽  
Set Up

A commercial crystallization robot has been modified for use in setting up sitting-drop vapor-diffusion crystallization experiments, and for setting up protein crystallization screensin situ. The primary aim of this effort is the automated screening of crystallization of integral membrane proteins in detergent-containing solutions. However, the results of this work are of general utility to robotic liquid-handling systems. Sources of error that can prevent the accurate dispensing and mixing of solutions have been identified, and include local environmental, machine-specific and solution conditions. Solutions to each of these problems have been developed and implemented.

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