WITHDRAWN: RhoB-dependent modulation of early endocytic traffic in Madin-Darby canine kidney cells.
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Journal Article↗
Biomedical subjects
Paul A Poland
Publications and source records attributed to Paul A Poland.
Recycling of MUC1 is dependent on its palmitoylation.
MUC1 is a mucin-like transmembrane protein expressed on the apical surface of epithelia, where it protects the cell surface. The cytoplasmic domain has numerous sites for phosphorylation and docking of proteins involved in signal transduction. In a previous study, we showed that the cytoplasmic YXXphi motif Y20HPM and the tyrosine-phosphorylated Y60TNP motif are required for MUC1 clathrin-mediated endocytosis through binding AP-2 and Grb2, respectively (Kinlough, C. L., Poland, P. A., Bruns, J. B., Harkleroad, K. L., and Hughey, R. P. (2004) J. Biol. Chem. 279, 53071-53077). Palmitoylation of transmembrane proteins can affect their membrane trafficking, and the MUC1 sequence CQC3RRK at the boundary of the transmembrane and cytoplasmic domains mimics reported site(s) of S-palmitoylation. [3H]Palmitate labeling of Chinese hamster ovary cells expressing MUC1 with mutations in CQC3RRK revealed that MUC1 is dually palmitoylated at the CQC motif independent of RRK. Lack of palmitoylation did not affect the cold detergent solubility profile of a chimera (Tac ectodomain and MUC1 transmembrane and cytoplasmic domains), the rate of chimera delivery to the cell surface, or its half-life. Calculation of rate constants for membrane trafficking of wild-type and mutant Tac-MUC1 indicated that the lack of palmitoylation blocked recycling, but not endocytosis, and caused the chimera to accumulate in a EGFP-Rab11-positive endosomal compartment. Mutations CQC/AQA and Y20N inhibited Tac-MUC1 co-immunoprecipitation with AP-1, although mutant Y20N had reduced rates of both endocytosis and recycling, but a normal subcellular distribution. The double mutant chimera AQA+Y20N had reduced endocytosis and recycling rates and accumulated in EGFP-Rab11-positive endosomes, indicating that palmitoylation is the dominant feature modulating MUC1 recycling from endosomes back to the plasma membrane.
Animals↗
Gamma-glutamyltranspeptidase: disulfide bridges, propeptide cleavage, and activation in the endoplasmic reticulum.
gamma-Glutamyltranspeptidase (gammaGT) is found primarily on the apical surface of epithelial and endothelial cells, where it degrades reduced and oxidized glutathione (gamma-GluCysGly) by hydrolysis of the unique gamma-glutamyl bond. Glutathione plays a key role in disulfide rearrangement in the endoplasmic reticulum (ER) and acts as a redox buffer. Previous work has shown that overexpression of gammaGT or an inactive splice variant gammaGTDelta7 mediates a redox stress response in the endoplasmic reticulum (ER) characterized by increased levels of BiP and induction of CHOP-10. To determine whether a CX(3)C motif might be the common feature of gammaGT and gammaGTDelta7 that mediates this response, we characterized disulfide bridges in gammaGT that might form between the six highly conserved Cys residues. Using site-directed mutagenesis of gammaGT, expression in Chinese Hamster Ovary (CHO) cells, metabolic labeling, and immunoblotting, our data predict disulfide formation between Cys49 and Cys73 and between Cys191 and Cys195 (the CX(3)C motif). Potential functions for this CX(3)C motif are discussed. In the course of defining the disulfides, we also noted that propeptide cleavage correlated with enzymatic activity. Because recent reports indicate that the homologous Escherichia coli gammaGT is a member of the N-terminal nucleophile (Ntn) hydrolase family, where the amino acid at the new N-terminus functions as the nucleophile for both autocatalytic cleavage and enzymatic activity, the rat gammaGT was similarly characterized. As predicted, mutations at the propeptide cleavage site coincidentally inhibit both heterodimer formation and gammaGT enzymatic activity. Analysis of early cleavage events using cell extraction into SDS indicates that propeptide cleavage occurs while gammaGT is still within the ER. Because activation and cleavage are coincident events, this raises the new question of whether an active glutathionase is present within the ER and what role gammaGT plays in modulating ER glutathione levels that are so critical for proper redox balance and disulfide formation in this compartment.
Animals↗
MUC1 membrane trafficking is modulated by multiple interactions.
MUC1 is a mucin-like transmembrane protein found on the apical surface of many epithelia. Because aberrant intracellular localization of MUC1 in tumor cells correlates with an aggressive tumor and a poor prognosis for the patient, experiments were designed to characterize the features that modulate MUC1 membrane trafficking. By following [(35)S]Met/Cys-labeled MUC1 in glycosylation-defective Chinese hamster ovary cells, we found previously that truncation of O-glycans on MUC1 inhibited its surface expression and stimulated its internalization by clathrin-mediated endocytosis. To identify signals for MUC1 internalization that are independent of its glycosylation state, the ectodomain of MUC1 was replaced with that of Tac, and chimera endocytosis was measured by the same protocol. Endocytosis of the chimera was significantly faster than for MUC1, indicating that features of the highly extended ectodomain inhibit MUC1 internalization. Analysis of truncation mutants and tyrosine mutants showed that Tyr(20) and Tyr(60) were both required for efficient endocytosis. Mutation of Tyr(20) significantly blocked coimmunoprecipitation of the chimera with AP-2, indicating that Y(20)HPM is recognized as a YXXphi motif by the mu2 subunit. The tyrosine-phosphorylated Y(60)TNP was previously identified as an SH2 site for Grb2 binding, and we found that mutation of Tyr(60) blocked coimmunoprecipitation of the chimera with Grb2. This is the first indication that Grb2 plays a significant role in the endocytosis of MUC1.
Adaptor Protein Complex 2↗
Maturation of the epithelial Na+ channel involves proteolytic processing of the alpha- and gamma-subunits.
The epithelial Na+ channel (ENaC) is a tetramer of two alpha-, one beta-, and one gamma-subunit, but little is known about its assembly and processing. Because co-expression of mouse ENaC subunits with three different carboxyl-terminal epitope tags produced an amiloride-sensitive sodium current in oocytes, these tagged subunits were expressed in both Chinese hamster ovary or Madin-Darby canine kidney type 1 epithelial cells for further study. When expressed alone alpha-(95 kDa), beta-(96 kDa), and gamma-subunits (93 kDa) each produced a single band on SDS gels by immunoblotting. However, co-expression of alphabetagammaENaC subunits revealed a second band for each subunit (65 kDa for alpha, 110 kDa for beta, and 75 kDa for gamma) that exhibited N-glycans that had been processed to complex type based on sensitivity to treatment with neuraminidase, resistance to cleavage by endoglycosidase H, and GalNAc-independent labeling with [3H]Gal in glycosylation-defective Chinese hamster ovary cells (ldlD). The smaller size of the processed alpha- and gamma-subunits is also consistent with proteolytic cleavage. By using alpha- and gamma-subunits with epitope tags at both the amino and carboxyl termini, proteolytic processing of the alpha- and gamma-subunits was confirmed by isolation of an additional epitope-tagged fragment from the amino terminus (30 kDa for alpha and 18 kDa for gamma) consistent with cleavage within the extracellular loop. The fragments remain stably associated with the channel as shown by immunoblotting of co-immunoprecipitates, suggesting that proteolytic cleavage represents maturation rather than degradation of the channel.
Animals↗