Cellular trafficking of phospholamban and formation of functional sarcoplasmic reticulum during myocyte differentiation

University of Illinois at Chicago, Chicago, Illinois, United States
AJP Cell Physiology (Impact Factor: 3.78). 07/2007; 292(6):C2084-94. DOI: 10.1152/ajpcell.00523.2006
Source: PubMed


Phospholamban (PLB) associates with the Ca(2+)-ATPase in sarcoplasmic reticulum (SR) membranes to permit the modulation of contraction in response to beta-adrenergic signaling. To understand how coordinated changes in the abundance and intracellular trafficking of PLB and the Ca(2+)-ATPase contribute to the maturation of functional muscle, we measured changes in abundance, location, and turnover of endogenous and tagged proteins in myoblasts and during their differentiation. We found that PLB is constitutively expressed in both myoblasts and differentiated myotubes, whereas abundance increases of the Ca(2+)-ATPase coincide with the formation of differentiated myotubes. We observed that PLB is primarily present in highly mobile vesicular structures outside the endoplasmic reticulum, irrespective of the expression of the Ca(2+)-ATPase, indicating that PLB targeting is regulated through vesicle trafficking. Moreover, using pulse-chase methods, we observed that in myoblasts, PLB is trafficked through directed transport through the Golgi to the plasma membrane before endosome-mediated internalization. The observed trafficking of PLB to the plasma membrane suggests an important role for PLB during muscle differentiation, which is distinct from its previously recognized role in the regulation of the Ca(2+)-ATPase.

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Available from: Scott T Brady, Oct 16, 2014
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    • "Because calmodulin effectively colocalizes with the L-type calcium channel [39], the results associated with the “single channel” volume scaling (Figure 5 and Section 2.2) are most relevant. Such colocalization is ubiquitous; many regulators have been shown to colocalize with channels or receptors, including phospholamban with calcium ATPase in the sarcoplasmic reticulum membrane [40], G-protein receptor kinases with G-protein receptors on the cell membrane [41], and Bax with voltage-dependent ion channels in the mitochondrial membrane [42]. Additionally, the volume of diadic subspaces can be greatly altered during pathophysiological conditions. "
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    • "PLN expression has been observed outside of the ER, suggesting that it is not exclusively contained within the ER and indicating a requirement for further cellular trafficking mechanisms [40]. In this study, we have established a possible mechanism for the trafficking of PLN. "
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    ABSTRACT: Phospholamban (PLN) is an effective inhibitor of the sarco(endo)plasmic reticulum Ca(2+)-ATPase, which transports Ca(2+) into the SR lumen, leading to muscle relaxation. A mutation of PLN in which one of the di-arginine residues at positions 13 and 14 was deleted led to a severe, early onset dilated cardiomyopathy. Here we were interested in determining the cellular mechanisms involved in this disease-causing mutation. Mutations deleting codons for either or both Arg13 or Arg14 resulted in the mislocalization of PLN from the ER. Our data show that PLN is recycled via the retrograde Golgi to ER membrane traffic pathway involving COP-I vesicles, since co-immunoprecipitation assays determined that COP I interactions are dependent on an intact di-arginine motif as PLN RDelta14 did not co-precipitate with COP I containing vesicles. Bioinformatic analysis determined that the di-arginine motif is present in the first 25 residues in a large number of all ER/SR Gene Ontology (GO) annotated proteins. Mutations in the di-arginine motif of the Sigma 1-type opioid receptor, the beta-subunit of the signal recognition particle receptor, and Sterol-O-acyltransferase, three proteins identified in our bioinformatic screen also caused mislocalization of these known ER-resident proteins. We conclude that PLN is enriched in the ER due to COP I-mediated transport that is dependent on its intact di-arginine motif and that the N-terminal di-arginine motif may act as a general ER retrieval sequence.
    PLoS ONE 07/2010; 5(7):e11496. DOI:10.1371/journal.pone.0011496 · 3.23 Impact Factor
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