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Vol. 19, Issue 7, 2777-2788, July 2008

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The Subcellular Distribution of Calnexin Is Mediated by PACS-2

Nathan Myhill^*, Emily M. Lynes^*, Jalal A. Nanji^*, Anastassia D. Blagoveshchenskaya^,, Hao Fei^,, Katia Carmine Simmen^,||, Timothy J. Cooper^*, Gary Thomas, and Thomas Simmen^*,

*Department of Cell Biology, University of Alberta, Edmonton, Alberta, T6G2H7, Canada; and Vollum Institute, Oregon Health and Science University, Portland, OR 97239

Submitted October 3, 2007; Revised March 28, 2008; Accepted April 9, 2008
Monitoring Editor: Adam Linstedt

Calnexin is an endoplasmic reticulum (ER) lectin that mediates protein folding on the rough ER. Calnexin also interacts with ER calcium pumps that localize to the mitochondria-associated membrane (MAM). Depending on ER homeostasis, varying amounts of calnexin target to the plasma membrane. However, no regulated sorting mechanism is so far known for calnexin. Our results now describe how the interaction of calnexin with the cytosolic sorting protein PACS-2 distributes calnexin between the rough ER, the MAM, and the plasma membrane. Under control conditions, more than 80% of calnexin localizes to the ER, with the majority on the MAM. PACS-2 knockdown disrupts the calnexin distribution within the ER and increases its levels on the cell surface. Phosphorylation by protein kinase CK2 of two calnexin cytosolic serines (Ser554/564) reduces calnexin binding to PACS-2. Consistent with this, a Ser554/564 Asp phosphomimic mutation partially reproduces PACS-2 knockdown by increasing the calnexin signal on the cell surface and reducing it on the MAM. PACS-2 knockdown does not reduce retention of other ER markers. Therefore, our results suggest that the phosphorylation state of the calnexin cytosolic domain and its interaction with PACS-2 sort this chaperone between domains of the ER and the plasma membrane.

Present addresses: ^||Department of Biochemistry, University of Alberta, Edmonton, AB, T6G 2H7, Canada;

Department of Psychiatry and Biobehavioral Sciences, The David Geffen School of Medicine at UCLA, Los Angeles, CA 90095;

Department of Medicine, Division of Nephrology and Hypertension, Oregon Health and Science University, Portland, OR 97239.

Address correspondence to: Thomas Simmen (Thomas.Simmen{at}ualberta.ca)

Abbreviations used: BAP31, B-cell receptor associated protein of 31 kDa; CI-MPR, cation-independent mannose 6-phosphate receptor; CK2, protein kinase CK2; CNX, calnexin; COPI, coatomer; ER, endoplasmic reticulum; ERK, extracellular signal regulated kinase; MAM, mitochondria-associated membrane; PACS-2, phosphofurin acidic cluster sorting protein 2; PDI, protein disulfide isomerase; rER, rough endoplasmic reticulum