190

Other Correspondence The methylation are of phosphatidylethanolamine in rat liver

microsomes

The letter from J. M. Mato x concerning incorporation of 14C methyl groups from S-adenosylmethionine into rat liver microsomal phosphatidylcholine indi- cates confusion between the results of experirnents in which the methyl- transferases are probed and experiments in which the products of methylation, monomethyl-PtdEth, dimethyl-PtdEth and PtdCho, are probed 2J. There is no dispute that trypsin treatment of micro- somes, under conditions in which these vesicles remain closed, inhibits incor- poration of methyl groups into PtdCho by 95% 2,4. However, the low level of ~4C methyl groups incorporated into the phospholipids of trypsin treated micro- somes either at high or low pH is mainly in monomethyl-PtdEth 2. Incorporation of ~4C methyl groups into this lipid is inhibited only 10% by trypsin treatment unless the microsomal vesicles are opened, when inhibition is increased to 75% 2. If the three methylation steps are closely linked, inhibition of either or

both of the second and third methylation steps would also inhibit the first meth- ylation step, as monomethyI-PtdEth accumulates. Thus, the observation that trypsin treatment of microsomes inhibits incorporation of ~C methyl groups into PtdCho 4 does not necessarily indicate that all three methylation steps occur at the outer surface of the membrane bilayer. Experiments in which the site of the products of methylation of PtdEth are probed using phospholipase C on trypsin treated and untreated micro- somes indicate that the product of the first methylation step is not in the outer leaflet of the membrane bilayer. Taken in conjunction with the results of experi- ments using trypsin, it appears that the first methylation step takes place at the inner leaflet of the membrane bilayer and subsequent steps in the outer leaf- let 2. This is also the conclusion from experiments of Hirata et al. on red blood cells 3 and rat liver microsomes s. We have not commented on the number of

T I B S - May 1985

methyltransferases involved in methyla- tion of PtdEth in rat liver microsomes, as our experiments were not designed to investigate this. However, as Dr Hirata 6 pointed out, the differential sensitivity of the methylation steps to trypsin and the different sites of the products of these steps do suggest that there may be more than one methyltransferase in rat liver microsomes.

References 1 Mato, J. M. (1984) Trends Biochem. Sci. 9,

515 2 Higgins, J. A. (1981) Biochlm. Biophys. Acta

640, 1-15 3 Hirata, F. and Axelrod, J. (1978) Proc. Natl

Acad. Sci. USA 75, 2348-2352 4 Audubert, F. and Vance, D. E. (1984) B/o-

chim. Biophys. Acta 792, 359-362 5 Sastry, R., Statham, J., Axelrod, J. and

Hirata, F. (1981) Arch. Biochem. Biophys. 211, 762-769

6 Hirata, F. (1984) Trends Biochem. Sci. 9, 514

JOAN A. HIGGINS

Department of Biochemistry, University of Sheffield, Sheffield, UK.

Receptosomes, endosomes, CURL: different terms for the same organelle system

In a recent letter to TIBS ~, Dr Kolb- Bachofen addresses two important issues: the nature of the vesicles we have termed receptosomes 2, and the mechan- ism of their formation.

The nature of receptosomes Receptosomes were described as hav-

ing characteristic features which should allow their identification whether or not they contain an electron-dense label or a ligand undergoing endocytosis 2,3. In the electron microscope, receptosomes are membrane-limited vesicles, ~ / ~ in diameter with an apparently empty center. They often have eccentrically located tubular protrusions and contain one or a few small circular membrane- limited inclusions; the latter have some- times been referred to as one type of 'multivesicular body'. Often one portion of a receptosome has a straightened edge that contains a fuzzy material on its cytoplasmic face (whether or not this material is related to clathrin is not clear). Receptosomes are, at least for part of their existence, isolated vesicles which in living cells can be seen to move by saltatory motion when the vesicles are labelled with a fluorescent ligand 4. Because of their small size and low den-

sity, receptosomes are not directly visible using phase-contrast or differen- tial interference-contrast microscopy.

In morphology, receptosomes are identical to structures termed 'endo- somes 's and resemble images of the structures termed 'CURL' 6. CURL was identified as the intracellular site where ligand and receptor uncoupled; pub- lished images of CURL resemble those of receptosomes in communication with tubules of what we have referred to as the trans-Golgi system. We have studied endocytosis in mouse, rat, hamster, dog and human fibroblastic cultured cells, in human epithelioid tumor cells (A431, KB), in human ovarian carcinoma ceils, in human lymphoma cells, in avian and amphibian nucleated erythrocytes and in rat hepatic sinusoidal endothelial cells in intact liver; all these ceil types had characteristic receptosomes. There is morphological evidence for recepto- somes in virtually all vertebrate cells. Therefore, there seems to be no basis for the suggestion ~ that receptosomes are only found in flat cells.

The mechanism of receptosome formation

As pointed out by Dr Kolb-

Bachofen ~, images of what ap[aear to be isolated coated vesicles 1400 A in diam- eter have been published by numerous investigators. Because we were con- cerned that such structures might be tan- gential sections of coated pits connected in an adjacent section to the cell surface, we developed new fixation and staining methods to study this TM. Our results with fibroblasts, KB ceils and hepatic sinusoi- dal endothelial cells indicate that all 1400/~ coated structures at the cell sur- face that are engaged in endocytosis are connected to the surface; some are directly connected and others are con- nected by long, narrow necks up to 10 000 A long a,9. Such long, narrow necks would be extremely difficult to detect with the fixation methods used by Dr Kolb-Bachofen. While we believe that receptosomes form directly from coated pits, the exact mechanism of for- mation is unclear. One suggestion was that the receptosome might form from the membrane of the neck of the pit; it is also possible that the coated pit which is connected to the surface by a narrow neck may swell to reach the size of a receptosome before it separates from the surface. In any case, our morphol- ogy studies do not support the sugges- tion that coated pits pinch off from the membrane directly as 1400 ]k diameter coated vesicles.