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

Abstract

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. INTRODUCTIONThe nuclear envelope consists of an inner and outer membrane. The outer nuclear membrane (ONM) is continuous with the endoplasmic reticulum (ER) and connects with the inner nuclear membrane (INM) at the nuclear pore membrane (POM; Lusk et al., 2007). Integral membrane proteins that reside in the INM are initially inserted into the endoplasmic reticulum (ER) membrane and move via the POM to the INM where they maintain a concentrated localization by binding to chromatin or tethering to the nuclear lamina (Powell and Burke, 1990;Soullam and Worman, 1993;Holmer and Worman, 2001;Ohba et al., 2004;Lusk et al., 2007;Schirmer and Foisner, 2007). Until recently, conventional wisdom held that the localization of proteins to either the ER membrane or the INM is mutually exclusive. For example, proteins that are found within the INM, such as LAP1, are not generally found throughout the ER/ONM; likewise, ER proteins such as HMG-CoA reductase are strictly localized to the ER (Wright et al., 1988;Powell and Burke, 1990;Deng and Hochstrasser, 2006). It was recently shown that a notable exception to this paradigm is the yeast E3 ubiquitin ligase, Doa10p. Doa10p was long known to reside in the ER membrane (Swanson et al., 2001;Kreft et al., 2006). However, recent data indicate that Doa10p exhibits dual steady-state localizations, residing and functioning in both the ER membrane where it mediates ER-associated degradation of membrane and secretory proteins, and in the INM where it mediates degradation of the nuclear transcription factor MAT␣2 (Deng and Hochstrasser, 2006).The present study documents another exception to the mutual exclusivity of ER membrane and INM localizati...

An Absence of Nuclear Lamins in Keratinocytes Leads to Ichthyosis, Defective Epidermal Barrier Function, and Intrusion of Nuclear Membranes and Endoplasmic Reticulum into the Nuclear Chromatin

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Impaired mechanical response of an EDMD mutation leads to motility phenotypes that are repaired by loss of prenylation

“…This modification causes the protein to become more hydrophobic, and is thought to facilitate the localization of lamin to the INM, as well as regulating protein-protein interactions. During the maturation process of lamins A, B1 and B2, the last 15 amino acids, including the farnesylated cysteine, are subsequently cleaved (Barrowman et al, 2008).…”

A-type lamins bind both hetero- and euchromatin, the latter being regulated by lamina-associated polypeptide 2 alpha

“…B-type lamins are tightly associated with the INM through post-translational addition of a hydrophobic farnesyl group to their C terminus (Gruenbaum and Foisner 2015). In contrast, lamin C is not farnesylated at all, and pre-lamin A is only transiently farnesylated, as a C-terminal peptide, including the farnesyl group, is removed in a final processing step (Sinensky et al 1994;Pendas et al 2002;Barrowman et al 2008). Thus, A-type lamins can dissociate from the membrane into the nucleoplasm, where they interact with the non-membrane-bound LAP2 isoform, LAP2alpha (Dechat et al 2000).…”