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...

BackgroundThe proteolytic maturation of the nuclear protein lamin A by the zinc metalloprotease ZMPSTE24 is critical for human health. The lamin A precursor, prelamin A, undergoes a multi-step maturation process that includes CAAX processing (farnesylation, proteolysis and carboxylmethylation of the C-terminal CAAX motif), followed by ZMPSTE24-mediated cleavage of the last 15 amino acids, including the modified C-terminus. Failure to cleave the prelamin A “tail”, due to mutations in either prelamin A or ZMPSTE24, results in a permanently prenylated form of prelamin A that underlies the premature aging disease Hutchinson-Gilford Progeria Syndrome (HGPS) and related progeroid disorders.Methodology/Principal FindingsHere we have investigated the features of the prelamin A substrate that are required for efficient cleavage by ZMPSTE24. We find that the C-terminal 41 amino acids of prelamin A contain sufficient context to allow cleavage of the tail by ZMPSTE24. We have identified several mutations in amino acids immediately surrounding the cleavage site (between Y646 and L647) that interfere with efficient cleavage of the prelamin A tail; these mutations include R644C, L648A and N650A, in addition to the previously reported L647R. Our data suggests that 9 of the 15 residues within the cleaved tail that lie immediately upstream of the CAAX motif are not critical for ZMPSTE24-mediated cleavage, as they can be replaced by the 9 amino acid HA epitope. However, duplication of the same 9 amino acids (to increase the distance between the prenyl group and the cleavage site) impairs the ability of ZMPSTE24 to cleave prelamin A.Conclusions/SignificanceOur data reveals amino acid preferences flanking the ZMPSTE24 cleavage site of prelamin A and suggests that spacing from the farnesyl-cysteine to the cleavage site is important for optimal ZMPSTE24 cleavage. These studies begin to elucidate the substrate requirements of an enzyme activity critical to human health and longevity.

NK cell maturation is critical for normal effector function and the innate immune response to tumors and pathogens. However, the molecular pathways that control NK cell maturation remain largely undefined. Here, we investigate the role of SPPL3, an intramembrane aspartyl protease, in murine NK cell biology. We find that deletion of SPPL3 in the hematopoietic system reduces numbers of peripheral NK cells, clearance of MHC Class I-deficient tumors in vivo, and cytotoxicity against tumor cells in vitro. This phenotype is concomitant with reduced numbers of CD27+CD11b+ and CD27−CD11b+ NK cells, indicating a requirement for SPPL3 in efficient NK cell maturation. NK cell-specific deletion of SPPL3 results in the same deficiencies, revealing a cell-autonomous role for SPPL3 in these processes. CRISPR/Cas9 genomic editing in murine zygotes was used to generate knock-in mice with a catalytically compromised SPPL3 D271A allele. Mice engineered to express only SPPL3 D271A in NK cells phenocopy mice deleted for SPPL3, indicating a requirement for SPPL3 protease activity in NK cell biology. Our results identify SPPL3 as a cell-autonomous molecular determinant of NK cell maturation and expand the role of intramembrane aspartyl proteases in innate immunity.

Natural killer (NK) cells play an essential role in tumor surveillance through direct tumor cell lysis. Signal peptide peptidase-like 3 (SPPL3) is a highly conserved intramembrane aspartyl protease, but very little is known about its functions in vivo. We developed an SPPL3 conditional knockout mouse and found that deletion of SPPL3 within the hematopoietic system using Vav1-iCre resulted in a peripheral defect in NK cell number. The reduced NK cell population was hypofunctional in tumor clearance in vivo and direct tumor lysis in vitro. Analysis of SPPL3-deficient NK cells revealed a substantial block in terminal maturation, at the CD27+CD11b- stage. Deletion of SPPL3 in NK cells by NKp46-iCre confirmed a cell-autonomous requirement for SPPL3 in NK development and function. CRISPR/Cas9 genome editing was used to create a knockin mouse expressing a protease-dead SPPL3 knockin allele, D271A. NK cells expressing SPPL3 D271A exhibited the same block in terminal differentiation as SPPL3-deficient NK cells. Our results reveal that the proteolytic function of the SPPL3 intramembrane protease is critical for normal NK cell maturation at a checkpoint that regulates the transition from CD11b- to CD11b+ cells.