Nucleoplasmic localization of prelamin A: implications for prenylation-dependent lamin A assembly into the nuclear lamina.

Abstract

The synthesis of the nuclear lamina protein lamin A requires the prenylation-dependent processing of its precursor protein, prelamin A. Unlike p2lr", which undergoes similar initial posttranslational modifications, maturation of lamin A results in the proteolytic removal of the prenylated portion of the molecule. We have used an in vitro prenylation system to demonstrate the nature of the prenyl substituent on prelamin A to be a farnesyl group. Further, the in vitro farnesylation of prelamin A requires an intact cysteinealiphatic-aliphatic-other (CAAX) amino acid sequence motif at its carboxyl terminus. The effect of blocking the prenylation of prelamin A on its localization and assembly into the nuclear lamina was investigated by indirect immunofluorescence. Expression of wild-type prelamin A in lovastatin-treated cells showed that nonprenylated prelamin A accumulated as nucleoplasmic particles. Upon addition of mevalonate to lovastatintreated cells, the wild-type lamin A was incorporated into the lamina within 3 hr. Expression of a mutant lamin A in which the carboxyl-terminal 21 amino acids were deleted resulted in a lamin molecule that was directly assembled into the lamina. These results indicate that the carboxyl-terminal peptide of prelamin A blocks its proper assembly into the nuclear lamina and that the prenylation-initiated removal of this peptide can occur in the nucleus.The nuclear lamina is a polymeric protein structure that lines the inner nuclear membrane. In most mammalian cells, it consists of three major proteins, lamins A-C (for review, see ref. 1). Mature lamin A is synthesized from a larger precursor protein (2, 3) and lacks the carboxyl-terminal 18 amino acids predicted by the cDNA sequence (4). Conversion ofthe lamin A precursor, prelamin A, to lamin A is dependent upon the isoprenylation of prelamin A (5). Prelamin A is an example of a class of proteins terminating in the sequence cysteinealiphatic-aliphatic-other (CAAX), which is prenylated at the consensus cysteine (for reviews, see refs. 6 and 7). This motif, shared by such proteins as p215 and the a-type mating factor of Saccharomyces, entrains a series of posttranslational processing steps. These include prenylation at the CAAX cysteine with either a 15-carbon (farnesyl) or 20-carbon (geranylgeranyl) isoprenoid followed by proteolytic removal of the A-A-X amino acids and carboxyl methylation of the now terminal cysteine. Evidence has been reported that the B-type lamins also undergo these prenylationdependent processing reactions (8-10). However, specific steps for the processing of prelamin A have not been directly demonstrated. Activities capable of catalyzing the carboxylterminal processing of a-factor and p2l's in vitro have been described (11,12). For p21'S, these activities were localized to the cytosolic and microsomal compartments (12, 13).Prelamin A and the yeast a-factor share an additional reaction subsequent to these carboxyl-terminal processing events. Both are subject to an endoproteolytic cleavage of their resp...

Analysis of Prelamin A Biogenesis Reveals the Nucleus to be a CaaX Processing Compartment

“…Our data reconciles the paradox surrounding nuclear lamin A processing, and indicates that the nucleus is a CaaXprocessing compartment, in addition to the ER membrane where Ras, Rho, a-factor, and other CaaX proteins are known to be modified (Gelb et al, 2006;Wright and Philips, 2006). Our findings provide a mechanistic and logistical basis for earlier evidence suggesting that the nuclear import of prelamin A via its strong NLS precedes its processing (Lehner et al, 1986;Lutz et al, 1992;Sasseville and Raymond, 1995). Confirmation of nuclear processing of lamin A also significantly impacts speculation of the as-yet-undetermined fate of the lamin A tail that is released by Zmpste24-mediated endoproteolytic cleavage ( Figure 1A, step 4).…”

“…Early evidence had suggested that prelamin A is processed within the nucleus (Lehner et al, 1986;Beck et al, 1990;Goldman et al, 1992;Lutz et al, 1992;Sasseville and Raymond, 1995). However, the postprenylation CaaX-processing enzymes Icmt, Zmpste24, and possibly Rce1 are known to be integral membrane ER-resident proteins with their active sites facing the cytosol, suggesting that prelamin A could be processed before nuclear import Schmidt et al, 1998;Corrigan et al, 2005).…”

“…For example, prelamin A could be exported out of the nucleus, processed at the ER, and reimported by diffusion in the membrane around the nuclear pore. Alternatively, it remains possible that the prelamin A within the nucleus may simply incorporate into the lamina in its unprocessed form, because the previous studies suggested, but did not confirm, maturation into lamin A (Lutz et al, 1992;Sasseville and Raymond, 1995).…”

“…For example, prelamin A could be exported out of the nucleus, processed at the ER, and reimported by diffusion in the membrane around the nuclear pore. Alternatively, it remains possible that the prelamin A within the nucleus may simply incorporate into the lamina in its unprocessed form, because the previous studies suggested, but did not confirm, maturation into lamin A (Lutz et al, 1992;Sasseville and Raymond, 1995).Here we have critically addressed the nuclear processing model and, as pointed to in previous studies, show that prelamin A can be entirely posttranslationally modified within the nuclear environment. Importantly, we demonstrate that this processing occurs without access to the cytosol or ER membranes.…”

“…However, other published data suggests that the entire processing pathway for prelamin A can occur within the nucleus. Notably, nucleoplasmic accumulation of completely unprocessed prelamin A has been observed under a variety of conditions that block farnesylation, the first step of CaaX processing (Lutz et al, 1992;Sasseville and Raymond, 1995). On reversal of this block, lamin A was observed at the nuclear rim.…”