Homeostatic restitution of cell membranes. Nuclear membrane lipid biogenesis and transport of protein from cytosol to intranuclear spaces.

Abstract

Our studies on homeostatic restitution of cellular and subcellular membranes showed that vesicular intracellular transport is engaged in systematic and coordinated replacement of lipids and proteins in the membranes of the secretory, non-dividing epithelial cells (Slomiany et al., J. Physiol. Pharmacol. 2004; 55: 837-860). In this report, we present evidence on the homeostatic restitution of lipids in the biomembranes that constitute nuclear envelopes. We investigated nuclear membranes lipid synthesis by employing purified intact nuclei (IN), the outer nuclear membrane (ONM), the inner nuclear membrane (INM) and the cell cytosol (CC). In contrast to Endoplasmic Reticulum (ER) which in the presence of CC generates new biomembrane that forms ER vesicles transporting ER products to Golgi, the IN, ONM and INM are not producing transport vesicles. Instead, the newly synthesized lipids remain in the nuclear membranes. The membranes (INM, ONM) of IN incubated with CC become enriched with newly synthesized phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylinositol phosphates (PIPs) and phosphatidic acid (PA). The incubation of separated ONM and INM with CC also enriched the membranes with IN specific lipids identified above. Moreover, the incubation of IN or its membranes with CC afforded retention of numerous CC proteins on the nuclear membrane. Here, we concentrated on 30kDa CC protein that displayed affinity to nuclear membrane PIP2. The 30kDa CC protein bound to PIP2 of IN, INM, and ONM. With IN, initially the PIP2-30kDa CC protein complex was detected on ONM, after 30-120 min of incubation, was found on INM and in nuclear contents. At the same time when the 30 kDa protein was released from INM and found in nuclear contents, the PIP2 of INM and ONM became undetectable, while the lipid extract from the membrane displaced from IN contained labeled PI only. Since ONM is an uninterrupted continuum of ER and INM, we speculate that the synthesis of the lipids in the ER, in the region adjacent to nucleus, is defining nuclear outer and inner biomembrane composition, is responsible for transport of the cytosolic protein into the nucleus and, replenishment of ER membrane used for vesicular transport.

Concluding Step in Cell Restitution Cycle: ER Transport Vesicles with Sphingolipids in the Outer Leaflet of the Membrane Restore Lysosomes

“…And, in the same construal of the metabolic cycle, the products of endocytosis, distinguished by their SM placement, are reflecting the composition of apical or basolateral membranes and are recognizable as genuine cell membrane-derived autophagocytes which are destined for the engulfment by lysosomal membrane and intralysosomal degradation. In keeping with our results-driven conclusions that all cell membranes are in continuous cycle of synthesis, replacement and degradation, the speculation with regard of the restitution of the ER and Golgi, the organelles most heavily involved in synthesis, modification, and trafficking of protein, is eminent but not through reprocessing of transport vesicles [14] As found out in the case of ER, the part of membrane adjacent to nuclear contents provides the link between nuclear pores, and as demonstrated with nuclear turnover of phosphoinositides, is in a constant lateral motion transporting cytosolic proteins to the nucleus, while in the same membrane, the lipids' synthesis-propelling motion delivers RNAs to cytosol [40] [41]. The reemerging from nucleus membrane must serve as the initial template that signals the translation of just produced and released mRNA and the integration of the product within co-synthesized organelle and/or membrane-specific lipids.…”

“…The cells were prepared from rat gastric mucosa and the liver as described previously [41] [46]- [50]. The single cells that were separated from larger debris with the aid of specific cell size nylon mesh, were centrifuged at 50 xg for 2 min, washed twice with the enzyme-free medium, twice with the Minimum Essential Medium (MEM), and counted in hemocytometer.…”

“…The single cells that were separated from larger debris with the aid of specific cell size nylon mesh, were centrifuged at 50 xg for 2 min, washed twice with the enzyme-free medium, twice with the Minimum Essential Medium (MEM), and counted in hemocytometer. Thus prepared cells were then incubated in MEM for 3 hours with or without radiolabel, then where indicated incubated for 30 min with 100 nM cycloheximide (CHX), and then used for preparation of nuclei [51], subcellular organelles, cell cytosol [41] …”

“…In our interpretation of the early nuclear events, the translational units assemble the systems that then, en bloc, restore organellar and cell membrane elements. This is accomplished through generation of the ER-initiated transport vesicles of specific composition that regenerate organellar and cell membrane components and functions [39]- [41]. The vesicles for organelles' restitution contain organelle-specific membrane lipids as well as cotranslationally intercalated proteins that are permanently incorporated into the membrane and en bloc delivered to the precise site, where they remain in place until portion of the organelles undergo another cycle of restitution that evokes autophagocytic dissection [42].…”

The Flow of Information from Nucleus to Golgi Is Contingent upon Nuclear Membrane Synthesis and Protraction of the Ceramide-Containing Membrane to Endoplasmic Reticulum

“…The carefully separated nuclei from cell organelles and the cell cytosol were subjected to treatments described earlier that affords mixture of membrane fragments commonly identified as outer and inner nuclear membranes (ONM, INM) [34]. To separate them into distinct fractions, the preparation of purified nuclear pellet recovered after centrifugation at 27,000 rpm in Beckman 45 Ti rotor for 1h was suspended at concentration of 2 mg protein/ml in a buffer consisting of 50 mM TIS-HCl pH 7.4, 0.25 M sucrose, 10 mM MgCl 2 , 1 mM DTT, 10 mg/ml leupeptin and 2 mM PMSF and then adjusted to 1% (w/v) with sodium citrate.…”