Association of prenylated proteins with the plasma membrane and the inner nuclear membrane is mediated by the same membrane-targeting motifs

Targeting of nuclear lamins to the inner nuclear envelope membrane requires a nuclear localization signal and CaaX motif-dependent posttranslational modifications, including isoprenylation and carboxyl methylation. These modifications, although necessary for membrane targeting, are not sufficient to mediate stable association with membranes. We show that two variants of lamin B3 (i.e., B3a and B3b) exist in Xenopus oocytes. They are encoded by two alternatively spliced, developmentally regulated mRNAs. The two lamin variants differ greatly in their membrane association in meiotically matured eggs. The presence of an extra cysteine residue (as a potential palmitoylation site) and a basic cluster in conjunction with the CaaX motif function as secondary targeting signals responsible for the stable membrane association of lamin B3b in Xenopus eggs. Moreover, transfection experiments with Green Fluorescent Protein lamin tail chimeras and with a Green Fluorescent Protein N-Ras chimera show that these secondary motifs are sufficient to target proteins to the inner nuclear membrane and/or the plasma membrane. Implications for the intracellular trafficking of doubly lipidated proteins are discussed.     

The CaaX motif is required for isoprenylation, carboxyl methylation, and nuclear membrane association of lamin B2

Recent evidence suggests that the conserved COOH-terminal CaaX motif of nuclear lamins may play a role in targeting newly synthesized proteins to the nuclear envelope. We have shown previously that in rabbit reticulocyte lysates the cysteine residue of the CaaX motif of chicken lamin B2 is necessary for incorporation of a derivative of mevalonic acid, the precursor of isoprenoids. Here we have analyzed the properties of normal and mutated forms of chicken lamin B2 stably expressed in mouse L cells. Mutation of the cysteine residue of the CaaX motif to alanine or introduction of a stop codon immediately after the cysteine residue was found to abolish both isoprenylation and carboxyl methylation of transfected lamin B2. Concomitantly, although nuclear import of the mutant lamin B2 proteins was preserved, their association with the inner nuclear membrane was severely impaired. From these results we conclude that the COOH-terminal CaaX motif is required for isoprenylation and carboxyl methylation of lamins in vivo, and that these modifications are important for association of B-type lamins with the nucleoplasmic surface of the inner nuclear membrane.     

The fates of chicken nuclear lamin proteins during mitosis: evidence for a reversible redistribution of lamin B2 between inner nuclear membrane and elements of the endoplasmic reticulum

In chicken, three structurally distinct nuclear lamin proteins have been described. According to their migration on two-dimensional gels, these proteins have been designated as lamins A, B1, and B2. To investigate the functional relationship between chicken lamins and their mammalian counterparts, we have examined here the state of individual chicken lamin proteins during mitosis. Current models proposing functional specializations of mammalian lamin subtypes are in fact largely based on the observation that during mitosis mammalian lamin B remains associated with membrane vesicles, whereas lamins A and C become freely soluble. Cell fractionation experiments combined with immunoblotting show that during mitosis both chicken lamins B1 and B2 remain associated with membranes, whereas lamin A exists in a soluble form. In situ immunoelectron microscopy carried out on mitotic cells also reveals membrane association of lamin B2, whereas the distribution of lamin A is random. From these results we conclude that both chicken lamins B1 and B2 may functionally resemble mammalian lamin B. Interestingly, immunolabeling of mitotic cells revealed an association of lamin B2 with extended membrane cisternae that resembled elements of the endoplasmic reticulum. Quantitatively, we found that all large endoplasmic reticulum-like membranes present in metaphase cells were decorated with lamin B2-specific antibodies. Given that labeling of these mitotic membranes was lower than labeling of interphase nuclear envelopes, it appears likely that during mitotic disassembly and reassembly of the nuclear envelope lamin B2 may reversibly distribute between the inner nuclear membrane and the endoplasmic reticulum.     

The role of CaaX-dependent modifications in membrane association of Xenopus nuclear lamin B3 during meiosis and the fate of B3 in transfected mitotic cells

Recent evidence shows that the COOH-terminal CaaX motif of lamins is necessary to target newly synthesized proteins to the nuclear envelope membranes. Isoprenylation at the CaaX-cysteine has been taken to explain the different fates of A- and B-type lamins during cell division. A-type lamins, which loose their isoprenylation shortly after incorporation into the lamina structure, become freely soluble upon mitotic nuclear envelope breakdown. Somatic B-type lamins, in contrast, are permanently isoprenylated and, although depolymerized during mitosis, remain associated with remnants of nuclear envelope membranes. However, Xenopus lamin B3, the major B-type lamin of amphibian oocytes and eggs, becomes soluble after nuclear envelope breakdown in meiotic metaphase. Here we show that Xenopus lamin B3 is permanently isoprenylated and carboxyl methylated in oocytes (interphase) and eggs (meiotic metaphase). When transfected into mouse L cells Xenopus lamin B3 is integrated into the host lamina and responds to cell cycle signals in a normal fashion. Notably, the ectopically expressed Xenopus lamin does not form heterooligomers with the endogenous lamins as revealed by a coprecipitation experiment with mitotic lamins. In contrast to the situation in amphibian eggs, a significant portion of lamin B3 remains associated with membranes during mitosis. We conclude from these data that the CaaX motif-mediated modifications, although necessary, are not sufficient for a stable association of lamins with membranes and that additional factors are involved in lamin-membrane binding.     

Head and/or CaaX domain deletions of lamin proteins disrupt preformed lamin A and C but not lamin B structure in mammalian cells

The nuclear lamina is an important determinant of nuclear architecture. Mutations in A-type but not B-type lamins cause a range of human genetic disorders, including muscular dystrophy. Dominant mutations in nuclear lamin proteins have been shown to disrupt a preformed lamina structure in Xenopus egg extracts. Here, a series of deletion mutations in lamins A and B1 were evaluated for their ability to disrupt lamina structure in Chinese hamster ovary cells. Deletions of either the lamin A "head" domain or the C-terminal CaaX domain formed intranuclear aggregates and resulted in the disruption of endogenous lamins A/C but not lamins B1/B2. By contrast, "head-less" lamin B1 localized to the nuclear rim with no detectable effect on endogenous lamins, whereas lamin B1 CaaX domain deletions formed intranuclear aggregates, disrupting endogenous lamins A/C but not lamins B1/B2. Filter binding assays revealed that a head/CaaX domain lamin B1 mutant interacted much more strongly with lamins A/C than with lamins B1/B2. Regulated induction of this mutant in stable cell lines resulted in the rapid elimination of all detectable lamin A protein, whereas lamin C was trapped in a soluble form within the intranuclear aggregates. In contrast to results in Xenopus egg extracts, dominant negative lamin B1 (but not lamin A) mutants trapped replication proteins involved in both the initiation and elongation phases of replication but did not effect cellular growth rates or the assembly of active replication centers. We conclude that elimination of the CaaX domain in lamin B1 and elimination of either the CaaX or head domain in lamin A constitute dominant mutations that can disrupt A-type but not B-type lamins, highlighting important differences in the way that A- and B-type lamins are integrated into the lamina.     

Regulated docking of nuclear membrane vesicles to vimentin filaments during mitosis

During mitosis, several types of intermediate-sized filaments (IFs) undergo an extensive remodelling in response to phosphorylation by cdc 2 and other protein kinases. However, unlike the nuclear lamins, the cytoplasmic IFs do not seem to follow a fixed disassembly stereotype and often retain their physical continuity without depolymerizing into soluble subunits. To investigate potential interactions between mitotically modified IFs and other cellular structures, we have examined prometaphase-arrested cells expressing the IF protein vimentin. We demonstrate here that vimentin filaments associate in situ and co-fractionate with a distinct population of mitotic vesicles. These vesicles carry on their surfaces nuclear lamin B, the inner nuclear membrane protein p58, and wheat germ agglutinin (WGA)-binding proteins. Consistent with a tight interaction between the IFs and the mitotic membranes, vimentin, nuclear lamin B, and a 180-kD WGA-binding protein are co-isolated when whole mitotic homogenates are incubated with anti-vimentin or anti-lamin B antibodies immobilized on magnetic beads. The vimentin-associated vesicles are essentially depleted of ER, Golgi and endosomal membrane proteins. The interaction of vimentin with lamin B-carrying membranes depends on phosphorylation and is weakened by dephosphorylation during nuclear reassembly in vitro. These observations reveal a novel interaction between IFs and cellular membranes and further suggest that the vimentin filaments may serve as a transient docking site for inner nuclear membrane vesicles during mitosis.     

Conservation of the gene structure and membrane-targeting signals of germ cell-specific lamin LIII in amphibians and fish

Targeting of nuclear lamins to the inner nuclear membrane requires CaaX motif-dependent posttranslational isoprenylation and carboxyl methylation. We previously have shown that two variants of lamin LIII (i.e., LIII and LIIIb) in amphibian oocytes are generated by alternative splicing and differ greatly in their membrane association. An extra cysteine residue (as a potential palmitoylation site) and a basic cluster in conjunction with the CaaX motif function as secondary targeting signals responsible for stable membrane association of lamin LIIIb. cDNA sequencing and genomic analysis of the zebrafish Danio rerio lamin LIII uncovers a remarkable conservation of the genomic organization and of the two secondary membrane-targeting signals in amphibians and fish. The expression pattern of lamin LIII genes is also conserved between amphibians and fish. Danio lamin LIII is expressed in diplotene oocytes. It is absent from male germ cells but is expressed in Sertoli cells of the testis. In addition, we provide sequence information of the entire coding sequence of zebrafish lamin A, which allows comparison of all major lamins from representatives of the four classes of vertebrates.     

Developmental changes in the organization of the nuclear lamina in mouse liver

We have compared the organization of the nuclear lamina in adult and fetal mouse liver. Western blot analysis of the expression of lamins with specific antibodies indicates that lamin B is expressed throughout liver development, unlike lamins A and C which are absent in fetal liver. Using [125I]lamin in blot binding assays, we have observed that lamin B binds to at least three membrane proteins (96, 54 and 34 kDa) and to lamins A and C in adult nuclear envelopes, but only to the 54 and 34 kDa proteins and lamin B itself in fetal nuclear envelopes, where lamin B appears to be hyperphosphorylated.     

A role for nuclear lamins in nuclear envelope assembly.

The molecular interactions responsible for nuclear envelope assembly after mitosis are not well understood. In this study, we demonstrate that a peptide consisting of the COOH-terminal domain of Xenopus lamin B3 (LB3T) prevents nuclear envelope assembly in Xenopus interphase extracts. Specifically, LB3T inhibits chromatin decondensation and blocks the formation of both the nuclear lamina-pore complex and nuclear membranes. Under these conditions, some vesicles bind to the peripheral regions of the chromatin. These "nonfusogenic" vesicles lack lamin B3 (LB3) and do not bind LB3T; however, "fusogenic" vesicles containing LB3 can bind LB3T, which blocks their association with chromatin and, subsequently, nuclear membrane assembly. LB3T also binds to chromatin in the absence of interphase extract, but only in the presence of purified LB3. Additionally, we show that LB3T inhibits normal lamin polymerization in vitro. These findings suggest that lamin polymerization is required for both chromatin decondensation and the binding of nuclear membrane precursors during the early stages of normal nuclear envelope assembly.     

Lamin B constitutes an intermediate filament attachment site at the nuclear envelope

We found that urea extraction of turkey erythrocyte nuclear envelopes abolished their ability to bind exogenous 125I-vimentin, while, at the same time, it removed the nuclear lamins from the membranes. After purification of the lamins from such urea extracts, a specific binding between isolated vimentin and lamin B, or a lamin A + B hetero- oligomer, was detected by affinity chromatography. Similar analysis revealed that the 6.6-kD vimentin tail piece was involved in this interaction. By other approaches (quantitative immunoprecipitation, rate zonal sedimentation, turbidometric assays) a substoichiometric lamin B-vimentin binding was determined under in vitro conditions. It was also observed that anti-lamin B antibodies but not other sera (anti- lamin A, anti-ankyrin, preimmune) were able to block 70% of the binding of 125I-vimentin to native, vimentin-depleted, nuclear envelopes. These data, which were confirmed by using rat liver nuclear lamins, indicate that intermediate filaments may be anchored directly to the nuclear lamina, providing a continuous network connecting the plasma membrane skeleton with the karyoskeleton of eukaryotic cells.     

SUN1 interacts with nuclear lamin A and cytoplasmic nesprins to provide a physical connection between the nuclear lamina and the cytoskeleton

Nuclear migration and positioning within cells are critical for many developmental processes and are governed by the cytoskeletal network. Although mechanisms of nuclear-cytoskeletal attachment are unclear, growing evidence links a novel family of nuclear envelope (NE) proteins that share a conserved C-terminal SUN (Sad1/UNC-84 homology) domain. Analysis of Caenorhabditis elegans mutants has implicated UNC-84 in actin-mediated nuclear positioning by regulating NE anchoring of a giant actin-binding protein, ANC-1. Here, we report the identification of SUN1 as a lamin A-binding protein in a yeast two-hybrid screen. We demonstrate that SUN1 is an integral membrane protein located at the inner nuclear membrane. While the N-terminal domain of SUN1 is responsible for detergent-resistant association with the nuclear lamina and lamin A binding, lamin A/C expression is not required for SUN1 NE localization. Furthermore, SUN1 does not interact with type B lamins, suggesting that NE localization is ensured by binding to an additional nuclear component(s), most likely chromatin. Importantly, we find that the luminal C-terminal domain of SUN1 interacts with the mammalian ANC-1 homologs nesprins 1 and 2 via their conserved KASH domain. Our data provide evidence of a physical nuclear-cytoskeletal connection that is likely to be a key mechanism in nuclear-cytoplasmic communication and regulation of nuclear position.     

Mislocalization of prelamin A Tyr646Phe mutant to the nuclear pore complex in human embryonic kidney 293 cells

Mature lamin A is formed after post-translational processing of prelamin A, which includes prenylation and carboxymethylation of cysteine 661 in the CaaX motif, followed by two proteolytic cleavages by zinc metalloprotease (ZMPSTE24). We expressed several prelamin A mutants, C661S (defective in prenylation), Y646F (designed to undergo prenylation but not second proteolytic cleavage), double mutant, Y646F/C661S and Y646X (mature lamin A), and the wild-type construct in human embryonic kidney (HEK-293) cells. Only the Y646F mutant co-localized with nuclear pore complex proteins, including Nup53 and Nup98, whereas the other mutants localized to the nuclear envelope rim. The cells expressing Y646F mutant also revealed abnormal nuclear morphology which was partially rescued with the farnesyl transferase inhibitors. These data suggest that the unprenylated prelamin A is not toxic to the cells. The toxicity of prenylated prelamin A may be due to its association and/or accumulation at the nuclear pore complex which could be partially reversed by farnesyl transferase inhibitors.     

Cytoskeletal proteins connecting intermediate filaments to cytoplasmic and nuclear periphery

Intermediate filaments (IFs), together with microtubules and microfilaments build up the cytoskeleton of most eukaryotic cells. Cytoplasmic IFs form a dense filament network radiating from the nucleus and extending to the plasma membrane. The association between the cytoplasmic and nuclear surfaces appears to provide a continuous link important for the organisation of the cytoplasm, for cellular communication, and possibly for the transport into and out of the nucleus. Cytoplasmic IFs approach the nuclear surface, thin fibrils seem to connect the IFs with the nuclear pore complexes and a direct interaction of cytoplasmic IFs with the nuclear lamin B has been observed by in vitro binding studies. However, none of the components that cross-link IFs to the nucleus has been unambiguously identified. Furthermore, if a direct interaction between cytoplasmic IFs and the nuclear lamin B occurs in vivo, the question of how cytoplasmic IFs get access to the nuclear interior remains to be resolved. The association of IFs with the plasma membranes involves different components, some of which are cell type specific. Two specialised complexes in epithelial cells: the desmosome and the hemidesmosome, serve as attachment sites for keratin filaments. Desmoplakin is considered as the cross-linking component of IFs to the desmosomal plaque, whereas BPAG1 (bullous pemphigoid antigen) would cross-link IFs at the hemidesmosomal plaque. In other cell types the modality of how IFs are anchored to the plasma membrane is less well understood. It involves different components such as the spectrin based membrane skeleton, ankyrin, myosin, plectin and certainly many other still unravelled partners. Association between the IFs and cellular membranes plays an important role in determining cell shape and tissue integrity. Thus, the identification and characterisation of the components involved in these interactions will be crucial for understanding the function of intermediate filaments.     

Type B lamins remain associated with the integral nuclear envelope protein p58 during mitosis: implications for nuclear reassembly.

p58 (also referred to as the lamin B receptor) is an integral membrane protein of the nuclear envelope known to form a multimeric complex with the lamins and other nuclear proteins during interphase. To examine the fate of this complex during mitosis, we have investigated the partitioning and the molecular interactions of p58 in dividing chicken hepatoma (DU249) cells. Using confocal microscopy and double immunolabelling, we show here that lamins B1 and B2 co-localize with p58 during all phases of mitosis and co-assemble around reforming nuclei. A close juxtaposition of p58/lamin B-containing vesicles and chromosomes is already detectable in metaphase; however, p58 and lamin reassembly proceeds slowly and is completed in late telophase--G1. Flotation of mitotic membranes in sucrose density gradients and analysis of mitotic vesicles by immunoelectron microscopy confirms that p58 and most of the type B lamins reside in the same compartment. Co-immunoprecipitation of both proteins by affinity-purified anti-p58 antibodies shows that they are physically associated in the context of a mitotic p58 'sub-complex'. This sub-assembly does not include the type A lamins which are fully solubilized during mitosis. Our data provide direct, in vivo and in vitro evidence that the majority of type B lamins remain connected to nuclear membrane 'receptors' during mitosis. The implications of these findings in nuclear envelope reassembly are discussed below.     

The lamin B receptor of the nuclear envelope inner membrane: a polytopic protein with eight potential transmembrane domains

The lamin B receptor is a previously identified integral membrane protein in the nuclear envelope of turkey erythrocytes that associates with the nuclear intermediate filament protein lamin B (Worman, H. J., J. Yuan, G. Blobel, and S. D. Georgatos. 1988. Proc. Natl. Acad. Sci. USA. 85:8531-8534). In the present report, we use cell fractionation and antibodies against the lamin B receptor to localize it to an 8-M urea-extracted membrane fraction of chicken liver nuclei, supporting an inner nuclear membrane localization. We deduced the amino acid sequence of the chicken lamin B receptor from overlapping clones obtained by screening cDNA libraries with a probe generated by the polymerase chain reaction with primers based on the partial protein sequence of the isolated protein. The mature lamin B receptor has a calculated molecular mass of 73,375 D and eight segments of hydrophobic amino acids that could function as transmembrane domains as determined by hydropathy analysis. Preceding the first putative transmembrane segment is a highly charged 204-residue-long amino terminal region that contains two consensus sites for phosphorylation by protein kinase A. Since the lamin B receptor has been shown to be phosphorylated by protein kinase A in vitro and in vivo and this phosphorylation affects lamin B binding (Applebaum, J., G. Blobel, and S. D. Georgatos. 1990. J. Biol. Chem. 265:4181-4185), it is likely that this amino terminal region faces the nucleoplasm. The amino terminal region also contains three DNA-binding motifs that are found in gene regulatory proteins and histones, suggesting that the lamin B receptor may additionally play a role in gene regulation and/or chromatin organization.     

Stepwise reassembly of the nuclear envelope at the end of mitosis

The nuclear envelope consists of three distinct membrane domains: the outer membrane with the bound ribosomes, the inner membrane with the bound lamina, and the pore membrane with the bound pore complexes. Using biochemical and morphological methods, we observed that the nuclear membranes of HeLa cells undergoing mitosis are disassembled in a domain-specific manner, i.e., integral membrane proteins representing the inner nuclear membrane (the lamin B receptor) and the nuclear pore membrane (gp210) are segregated into different populations of mitotic vesicles. At the completion of mitosis, the inner nuclear membrane- derived vesicles associate with chromatin first, beginning in anaphase, whereas the pore membranes and the lamina assemble later, during telophase and cytokinesis. Our data suggest that the ordered reassembly of the nuclear envelope is triggered by the early attachment of inner nuclear membrane-derived vesicles to the chromatin.     

Monitoring the disruption of nuclear envelopes in interphase cells with GFP-beta-galactosidase.

The nuclear envelope of eukaryotic cells provides a barrier separating nucleus from cytoplasm, thereby regulating the exchange of macromolecules between both compartments. However, in cells exposed to severe forms of stress this barrier may break down, resulting in the mixing of nuclear and cytoplasmic contents. We show here that the fusion protein GFP-beta-galactosidase can be used to evaluate the intactness of nuclear envelopes in HeLa cells that have been exposed to heat and oxidative stress. GFP-beta-galactosidase is restricted to the cytoplasm of interphase cells, but enters the nucleus when nuclear membranes are disrupted. For comparison, we have analyzed the barrier function of nuclear membranes with antibodies against lamin B. Treatment of fixed cells with digitonin permeabilizes the plasma membrane, but leaves nuclear envelopes intact. Consequently, after digitonin incubation antibodies to lamin B can bind their antigen only if nuclear membranes are damaged. For various heat and oxidative stress conditions, we have compared the distribution of GFP-beta-galactosidase with the accessibility of lamin B to antibodies. Our results demonstrate that nuclear envelopes are permeable to antibodies whenever GFP-beta-galactosidase enters the nucleus. GFP-beta-galactosidase is therefore a useful tool for evaluating the disintegration of the nuclear envelope and identifying cells in which a mixing of nuclear and cytoplasmic material takes place.     

Analysis of prelamin A biogenesis reveals the nucleus to be a CaaX processing compartment

Proteins establish and maintain a distinct intracellular localization by means of targeting, retention, and retrieval signals, ensuring most proteins reside predominantly in one cellular location. The enzymes involved in the maturation of lamin A present a challenge to this paradigm. Lamin A is first synthesized as a 74-kDa precursor, prelamin A, with a C-terminal CaaX motif and undergoes a series of posttranslational modifications including CaaX processing (farnesylation, aaX cleavage and carboxylmethylation), followed by endoproteolytic cleavage by Zmpste24. Failure to cleave prelamin A results in progeria and related premature aging disorders. Evidence suggests prelamin A is imported directly into the nucleus where it is processed. Paradoxically, the processing enzymes have been shown to reside in the cytosol (farnesyltransferase), or are ER membrane proteins (Zmpste24, Rce1, and Icmt) with their active sites facing the cytosol. Here we have reexamined the cellular site of prelamin A processing, and show that the mammalian and yeast processing enzymes Zmpste24 and Icmt exhibit a dual localization to the inner nuclear membrane, as well as the ER membrane. Our findings reveal the nucleus to be a physiologically relevant location for CaaX processing, and provide insight into the biology of a protein at the center of devastating progeroid diseases.     

Crystal structure of the human lamin A coil 2B dimer: implications for the head-to-tail association of nuclear lamins

Nuclear intermediate filaments (IFs) are made from fibrous proteins termed lamins that assemble, in association with several transmembrane proteins of the inner nuclear membrane and an unknown number of chromatin proteins, into a filamentous scaffold called the nuclear lamina. In man, three types of lamins with significant sequence identity, i.e. lamin A/C, lamin B1 and B2, are expressed. The molecular characteristics of the filaments they form and the details of the assembly mechanism are still largely unknown. Here we report the crystal structure of the coiled-coil dimer from the second half of coil 2 from human lamin A at 2.2A resolution. Comparison to the recently solved structure of the homologous segment of human vimentin reveals a similar overall structure but a different distribution of charged residues and a different pattern of intra- and interhelical salt bridges. These features may explain, at least in part, the differences observed between the lamin and vimentin assembly pathways. Employing a modeled lamin A coil 1A dimer, we propose that the head-to-tail association of two lamin dimers involves strong electrostatic attractions of distinct clusters of negative charge located on the opposite ends of the rod domain with arginine clusters in the head domain and the first segment of the tail domain. Moreover, lamin A mutations, including several in coil 2B, have been associated with human laminopathies. Based on our data most of these mutations are unlikely to alter the structure of the dimer but may affect essential molecular interactions occurring in later stages of filament assembly and lamina formation.     

Prenylated prelamin A interacts with Narf, a novel nuclear protein.

Prelamin A is farnesylated and methylated on the cysteine residue of a carboxyl-terminal CaaX motif. In the nucleus, prelamin A is processed to lamin A by endoproteolytic removal of the final 18 amino acids, including the farnesylated cysteine residue. Using the yeast two-hybrid assay, we isolated a novel human protein, Narf, that binds the carboxyl-terminal tail of prelamin A. Narf has limited homology to iron-only bacterial hydrogenases and eukaryotic proteins of unknown function. Narf is encoded by a 2-kilobase mRNA expressed in all human cell lines and tissues examined. The protein is detected in the nuclear fraction of HeLa cell lysates on Western blots and can be extracted from nuclear envelopes with 0.5 M NaCl. When a FLAG epitope-tagged Narf is expressed in HeLa cells, it is exclusively nuclear and partially co-localizes with the nuclear lamina. The farnesylation status of prelamin A determines its ability to bind to Narf. Inhibition of farnesyltransferase and mutation or deletion of the CaaX motif from the prelamin A tail domain inhibits Narf binding in yeast two-hybrid and in vitro binding assays. The prenyl-dependent binding of Narf to prelamin A is an important first step in understanding the functional significance of the lamin A precursor.