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A new class of Paramecium surface proteins anchored in the plasma membrane by a glycosylinositol phospholipid. Membrane anchor of Paramecium cross-reacting glycoproteins.

ABSTRACT: Treatment of paramecia with ethanol or Triton X-100 solubilizes a major membrane protein, namely the surface antigen (SAg), and a set of glycopeptides in the range 40-60 kDa, which cross-react with the SAg. We demonstrate that these glycopeptides, called 'cross-reacting glycoproteins' (CRGs), are distinct molecules from the SAg. First, after purification of CRGs from ethanolic extracts of Paramecium primaurelia expressing the 156G SAg, the amino acid composition of a given CRG was found to be different from, and incompatible with, that of the 156G SAg. Secondly, we showed that the CRGs, although not immunologically detectable, are present in fractions containing the myristoylated form of the 156G SAg. The treatment of these fractions by phosphatidylinositol-specific phospholipases C enables us to reveal the CRGs through the unmasking of two distinct epitopes. One is the 'cross-reacting determinant' (CRD), initially described for the variant surface glycoproteins (VSGs) of Trypanosoma; the other determinant, called 'det-2355', is specific to the SAg and to the CRGs. Our results suggest that (1) phosphatidylinositol is covalently linked to the CRGs and (2) the CRD and the det-2355 are localized in the same region of the CRGs. We propose that the CRGs are a new set of surface proteins anchored in the cell membrane of Paramecium via a glycosylinositol phospholipid, in the same way as the SAgs.

SUBMITTER: Deregnaucourt C

PROVIDER: S-EPMC1149312 | biostudies-other | 1988 Jul

REPOSITORIES: biostudies-other

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Project description:Tether proteins attach the endoplasmic reticulum (ER) to other cellular membranes, thereby creating contact sites that are proposed to form platforms for regulating lipid homeostasis and facilitating non-vesicular lipid exchange. Sterols are synthesized in the ER and transported by non-vesicular mechanisms to the plasma membrane (PM), where they represent almost half of all PM lipids and contribute critically to the barrier function of the PM. To determine whether contact sites are important for both sterol exchange between the ER and PM and intermembrane regulation of lipid metabolism, we generated ?-super-tether (?-s-tether) yeast cells that lack six previously identified tethering proteins (yeast extended synatotagmin [E-Syt], vesicle-associated membrane protein [VAMP]-associated protein [VAP], and TMEM16-anoctamin homologues) as well as the presumptive tether Ice2. Despite the lack of ER-PM contacts in these cells, ER-PM sterol exchange is robust, indicating that the sterol transport machinery is either absent from or not uniquely located at contact sites. Unexpectedly, we found that the transport of exogenously supplied sterol to the ER occurs more slowly in ?-s-tether cells than in wild-type (WT) cells. We pinpointed this defect to changes in sterol organization and transbilayer movement within the PM bilayer caused by phospholipid dysregulation, evinced by changes in the abundance and organization of PM lipids. Indeed, deletion of either OSH4, which encodes a sterol/phosphatidylinositol-4-phosphate (PI4P) exchange protein, or SAC1, which encodes a PI4P phosphatase, caused synthetic lethality in ?-s-tether cells due to disruptions in redundant PI4P and phospholipid regulatory pathways. The growth defect of ?-s-tether cells was rescued with an artificial "ER-PM staple," a tether assembled from unrelated non-yeast protein domains, indicating that endogenous tether proteins have nonspecific bridging functions. Finally, we discovered that sterols play a role in regulating ER-PM contact site formation. In sterol-depleted cells, levels of the yeast E-Syt tether Tcb3 were induced and ER-PM contact increased dramatically. These results support a model in which ER-PM contact sites provide a nexus for coordinating the complex interrelationship between sterols, sphingolipids, and phospholipids that maintain PM composition and integrity.

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A new class of Paramecium surface proteins anchored in the plasma membrane by a glycosylinositol phospholipid. Membrane anchor of Paramecium cross-reacting glycoproteins.

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