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CELL BIOLOGY AND SIGNAL TRANSDUCTION

Peroxisomal Ascorbate Peroxidase Resides within a Subdomain of Rough Endoplasmic Reticulum in Wild-Type Arabidopsis Cells¹

Department of Plant Biology and Graduate Program in Molecular and Cellular Biology, Arizona State University, Tempe, Arizona 85287–1601

Previously we reported (R.T. Mullen, C.S. Lisenbee, J.A. Miernyk, R.N. Trelease [1999] Plant Cell 11: 2167–2185) that overexpressed ascorbate peroxidase (APX), a peroxisomal membrane protein, sorted indirectly to Bright Yellow-2 cell peroxisomes via a subdomain of the endoplasmic reticulum (ER; peroxisomal endoplasmic reticulum [pER]). More recently, a pER-like compartment also was identified in pumpkin (Cucurbita pepo) and transformed Arabidopsis cells (K. Nito, K. Yamaguchi, M. Kondo, M. Hayashi, M. Nishimura [2001] Plant Cell Physiol 42: 20–27). Here, we characterize more extensively the localization of endogenous Arabidopsis peroxisomal APX (AtAPX) in cultured wild-type Arabidopsis cells (Arabidopsis var. Landsberg erecta). AtAPX was detected in peroxisomes, but not in an ER subcompartment, using immunofluorescence microscopy. However, AtAPX was detected readily with immunoblots in both peroxisomal and ER fractions recovered from sucrose (Suc) density gradients. Most AtAPX in microsomes (200,000g, 1 h pellet) applied to gradients exhibited a Mg²⁺-induced shift from a distribution throughout gradients (approximately 18%–40% [w/w] Suc) to ≥42% (w/w) Suc regions of gradients, including pellets, indicative of localization in rough ER vesicles. Immunogold electron microscopy of the latter fractions verified these findings. Further analyses of peroxisomal and rough ER vesicle fractions revealed that AtAPX in both fractions was similarly associated with and located mostly on the cytosolic face of the membranes. Thus, at the steady state, endogenous peroxisomal AtAPX resides at different levels in rough ER and peroxisomes. Collectively, these findings show that rather than being a transiently induced sorting compartment formed in response to overexpressed peroxisomal APX, portions of rough ER (pER) in wild-type cells serve as a constitutive sorting compartment likely involved in posttranslational routing of constitutively synthesized peroxisomal APX.

¹ This work was supported by the National Science Foundation (grant no. MCB–0091826 to R.N.T.) and in part by the William N. and Myriam Pennington Foundation. The Graduate Program in Molecular and Cellular Biology at Arizona State University funded a Research Assistantship for C.S.L.

² Present address: Mayo Clinic Scottsdale, Scottsdale, AZ 85259.

^* Corresponding author; e-mail trelease.dick{at}asu.edu; fax 480-965-6899.

Received January 3, 2003; returned for revision February 18, 2003; accepted March 26, 2003.

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