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

Abstract

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.

ArabidopsisPEROXIN11c-e, FISSION1b, and DYNAMIN-RELATED PROTEIN3A Cooperate in Cell Cycle–Associated Replication of Peroxisomes

“…In these cells, myc-FIS1b consistently colocalized with catalase within a few relatively large cytoplasmic structures ( Figure 1D, arrowheads), distinctly different in size and number from the individual normalappearing peroxisomes visible in the neighboring nontransformed cells ( Figure 1D, arrows) or in mock-transformed cells ( Figure 1H) or from the elongated peroxisomes observed in cells overexpressing myc-PEX11d alone ( Figure 1G). The abnormally large structures in Figure 1D, possessing catalase, myc-FIS1b, and PEX11d likely resulted from peroxisome aggregation, as we previously observed in cells overexpressing peroxisome membrane proteins (Mullen et al, 2001;Lisenbee et al, 2003;Murphy et al, 2003;McCartney et al, 2005). In parallel experiments, coexpressions of myc-FIS1b with untagged PEX11e also resulted in the apparent recruitment of myc-FIS1b to aggregated peroxisomes.…”

“…Immunolabeling of protoplasts was done as described previously (Lisenbee et al, 2003;Flynn et al, 2005) with the following modifications. Briefly, 2 to 4 d after transformation, formaldehyde-fixed protoplasts (see above) were adhered to poly-L-lysine-coated slides for 30 min, incubated in 0.33% (v/v) Triton X-100 in PBS for 15 min, and washed twice in PBS.…”

Pex3p Initiates the Formation of a Preperoxisomal Compartment from a Subdomain of the Endoplasmic Reticulum in Saccharomyces cerevisiae

“…In the yeast Yarrowia lipolytica, the peroxisomal membrane proteins Pex2p and Pex16p were shown to traffic through the ER and to acquire core N-linked glycosylation (7). Findings supporting de novo peroxisome biogenesis in close association with the ER were obtained in cells of Y. lipolytica temperature-sensitive for Pex3p function (8), and studies in the plant Arabidopsis showed that peroxisomal ascorbate peroxidase localized to a subdomain of rough ER that could serve as a compartment for posttranslational sorting to peroxisomes (9). In mouse dendritic cells, the peroxisomal membrane proteins Pex13p and PMP70 were found in subdomains of the ER that extended to a peroxisomal reticulum from which mature peroxisome arose (10).…”

Plant Peroxisomes: Biogenesis and Function

“…Another important, but poorly characterized, aspect of the ER-peroxisome relationship in plants is the nature of the peroxisomal ER (pER) subdomain, a region of the ER at which preperoxisomes are proposed to be formed (Mullen et al, 1999;Lisenbee et al, 2003). The PMPs APX3 and Arabidopsis PEX10 localize to subdomains of the rough ER (Lisenbee et al, 2003;Flynn et al, 2005;Trelease, 2005, 2007).…”

“…The PMPs APX3 and Arabidopsis PEX10 localize to subdomains of the rough ER (Lisenbee et al, 2003;Flynn et al, 2005;Trelease, 2005, 2007). However, whether these regions are identical and how the intra-ER sorting and segregation of APX and PEX10 (or any other PMP in the ER) is accomplished has not been elucidated.…”