Localization of mitochondrial DNA encoded cytochrome c oxidase subunits I and II in rat pancreatic zymogen granules and pituitary growth hormone granules

Abstract

Cytochrome c oxidase (COX) complex is an integral part of the electron transport chain. Three subunits of this complex (COX I, COX II and COX III) are encoded by mitochondrial (mit-) DNA. High-resolution immunogold electron microscopy has been used to study the subcellular localization of COX I and COX II in rat tissue sections, embedded in LR Gold resin, using monoclonal antibodies for these proteins. Immunofluorescence labeling of BS-C-1 monkey kidney cells with these antibodies showed characteristic mitochondrial labeling. In immunogold labeling studies, the COX I and COX II antibodies showed strong and specific mitochondrial labeling in the liver, kidney, heart and pancreas. However, in rat pancreatic acinar tissue, in addition to mitochondrial labeling, strong and specific labeling was also observed in the zymogen granules (ZGs). In the anterior pituitary, strong labeling with these antibodies was seen in the growth hormone secretory granules. In contrast to these compartments, the COX I or COX II antibodies showed only minimal labeling (five- to tenfold lower) of the cytoplasm, endoplasmic reticulum and the nucleus. Strong labeling with the COX I or COX II antibodies was also observed in highly purified ZGs from bovine pancreas. The observed labeling, in all cases, was completely abolished upon omission of the primary antibodies. These results provide evidence that, similar to a number of other recently studied mit-proteins, COX I and COX II are also present outside the mitochondria. The presence of mit-DNA encoded COX I and COX II in extramitochondrial compartments, provides strong evidence that proteins can exit, or are exported, from the mitochondria. Although the mechanisms responsible for protein exit/export remain to be elucidated, these results raise fundamental questions concerning the roles of mitochondria and mitochondrial proteins in diverse cellular processes in different compartments.

Subcellular localization of fumarase in mammalian cells and tissues

“…In earlier work, although many mitochondrial proteins have been localized in diVerent types of secretory granules (e.g. ZGs, GHGs and insulin secretory granules) as well as on plasma membrane, in most cases no clear evidence that signiWcant amounts of these proteins were also present in the ER lumen was obtained Soltys et al 2001;Sadacharan et al 2001Sadacharan et al , 2005. This has been partly due to the diYculty in clearly distinguishing the low amount of labeling seen in the ER/cytoplasm between the two compartments.…”

“…6a). Antibodies to a number of other mit-proteins, including cytochrome oxidase Table 1 subunits I and II, P32, Hsp60 and Cpn10 have previously been shown to show strong and speciWc labeling of ZGs Soltys et al 2001;Sadacharan et al 2001Sadacharan et al , 2005. With the fumarase antibody, we also observed some reactivity in the ER, but due to the relatively large size of the gold particles (20 nm) that were used in these experiments, we were not able to determine whether the labeling was speciWcally localized to the ER rather than in the cytoplasm.…”

“…However, in our earlier work with other mitochondrial proteins such as Hsp60, COX I and II, Cyt C, Cpn10, P32 etc. which were found to label ZGs, no clear evidence of labeling over ER was observed Soltys et al 2001;Sadacharan et al 2001Sadacharan et al , 2005. A control QuantiWcation of immunogold labeling with 20 nm gold particles was carried out as described in the Materials and methods section.…”

“…After the Wnal puriWcation step, an aliquot of the puriWed ZG preparation was Wxed in 4% paraformaldehyde, postWxed in uranyl acetate and after dehydration then embedded in LR white resin as described in earlier work (Soltys and Gupta 1996;Sadacharan et al 2005). The purity of the ZGs was assessed visually by means of electron microscopy and mitochondrial contamination in this fraction was determined to be less <2% .…”

“…(Velez-Granell et al 1994;Soltys and Gupta 1996;Singh et al 1997;Meertens et al 1998;Lakshmipathy and Campbell 1999;Cechetto et al , 2002Ran et al 2000;Felts et al 2000;Kotti et al 2000;Soltys et al 2001;Ghebrehiwet et al 2001;Sadacharan et al 2001;Brokstad et al 2001;Valgardsdottir et al 2001;Strobel et al 2002;Hubner et al 2003;Haraguchi et al 2003;Meenakshi et al 2003;Gingrich et al 2004;Yang et al 2004) and mit-DNA encoded (viz. NADH dehydrogenease subunits 1 and 2, cytochrome c oxidase (COX) subunits I and II and ATP synthase subunit 6) (Loveland et al 1990;Gingrich et al 2004;Sadacharan et al 2005), which are present at extramitochondrial locations have been discovered. A number of these proteins carry out unique functions at these ectopic locations which are diVerent from those in mitochondria (Loveland et al 1990;Isola et al 1995;Cavanagh 1996;Bradbury and Berk 2000;Ran et al 2000;Gingrich et al 2004;Gupta et al 2005).…”