Measurement of Heme Efflux and Heme Content in Isolated Developing Chloroplasts

Abstract

Hemes destined for cytosolic hemoproteins must originate in one of the cellular compartments which have the capacity for heme synthesis, namely the chloroplast or the mitochondria. Since developing chloroplasts from greening cucumber (Cucumis sativus, cv. Sumter) cotyledons are known to contain complete heme and chlorophyll biosynthetic pathways, they were tested for their capacity export hemes. Picomole quantities of heme were measured by reconstitution of the heme with apo-peroxidase and subsequent determination of peroxidase activity. The assay method was sensitive (as little as 0.7 picomole of heme could be detected in a volume of 100 microliters) and was linear with heme concentration. When intact plastids were incubated with apo-peroxidase, a steady-state rate of efflux between 0.12 and 0.45 picomole heme/minute/milligram plastid protein was measured. The efflux rate was not due to plastid breakage and could be enhanced by incubating with the heme precursor, baminolevulinic acid. Cold acetone extraction removed 47 ± 17 picomoles heme/milligram plastid protein from the total b-type heme pool in the chloroplasts (166 ± 9 picomoles heme/milligram protein, by acid-acetone extraction). The reconstitution technique provided a similar estimate of readily exchangeable heme in the plastid, 37 ± 8 picomoles heme/milligram protein (or 6 micromolar in the plastids). These values may be indicative of a 'free heme pool' which exists in the chloroplast.

Mechanism and regulation of Mg-chelatase

“…There is a good physiological argument that this may be the case : mitochondria cannot synthesize haem, although they do contain ferrochelatase, and therefore chloroplasts must supply either haem or haem precursors to mitochondria and the rest of the cell. The efflux of haem and other porphyrins from isolated chloroplasts has been observed [61][62][63] . Possibly there are two haem-synthesis pathways in the chloroplast : one for photosynthetic uses and the other for non-photosynthetic and\or extrachloroplastidic haem.…”

The Regulation of Enzymes Involved in Chlorophyll Biosynthesis

“…However, another part of protoheme is likely to be released from the thylakoids to the stroma, where the pigment becomes attached to either soluble c-type cytochromes (Bhaya and Castelfranco, 1985), or to other proteins, such as catalase (Dailey, 1990). In addition, protoheme export from the plastids to the cytosol has been reported (Thomas and Weinstein, 1990).…”

“…The identification of most of the aforementioned pigments and of many of the enzymes necessary for converting protoporphyrinogen IX to Pchlide in isolated envelope membranes of spinach chloroplasts seems to confirm this proposal. In addition, intermediates and products of the C, pathway, such as protoporphyrinogen IX (Lehnen et al, 1990), phytochromobilin (Terry and Lagarias, 1991) and (proto)heme (Thomas and Weinstein, 1990), have been demonstrated to be exportable from the plastids to the cytosol. One may therefore conclude that other intermediates of the C , pathway, such as Pchlide, might also be able to leave the plastid, and that either the pigments themselves or pigment-protein complexes could act either directly or indirectly, e.g.…”

“…Protoheme can either be incorporated into the plastidic hemoproteins, such as cytochromes f and b, of the cytochrome b$complex (Phillips and Gray, 1983;Merchant, 1992,1995;Huang et al, 1994), or be exported from the plastids to the cytosol (Thomas and Weinstein, 1990), where it serves different functions (Dailey, 1990). It is not known whether exclusively the plastidderived protoheme, or an earlier plastid precursor of protoheme, such as protoporphyrinogen IX, can be transported into mitochondria (Beale and Jacobs andJacobs, 1984, 1993).…”

Further Characterization of the Magnesium Chelatase in Isolated Developing Cucumber Chloroplasts

“…Intact chloroplasts were isolated from greening cucumber cotyledons as previously described (26), except that the final pellet was resuspended in grinding buffer (0.5 M sorbitol, 50 mM Tricine, 1 Chlorophyllide was generated by the degradation of Chl after the endogenous chlorophyllase was activated by treatment of the chloroplasts with 40% (v/v) acetone (4). The chlorophyllide was recovered by extraction into ether (7).…”