Prelamin A endoproteolytic processing in vitro by recombinant Zmpste24

Author:

CORRIGAN Douglas P.1,KUSZCZAK Danuta1,RUSINOL Antonio E.1,THEWKE Douglas P.1,HRYCYNA Christine A.2,MICHAELIS Susan3,SINENSKY Michael S.1

Affiliation:

1. Department of Biochemistry and Molecular Biology, James H. Quillen College of Medicine, East Tennessee State University, Box 70581, Johnson City, TN 37614-0581, U.S.A.

2. Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, IN 47907-2084, U.S.A.

3. Department of Cell Biology, The Johns Hopkins University School of Medicine, 725 N Wolfe St., Baltimore, MD 21205, U.S.A.

Abstract

The nuclear lamins form a karyoskeleton providing structural rigidity to the nucleus. One member of the lamin family, lamin A, is first synthesized as a 74 kDa precursor, prelamin A. After the endopeptidase and methylation reactions which occur after farnesylation of the CAAX-box cysteine, there is a second endoproteolysis that occurs 15 amino acids upstream from the C-terminal farnesylated cysteine residue. Studies with knockout mice have implicated the enzyme Zmpste24 (Face-1) as a suitable candidate to perform one or both of these proteolytic reactions. Evidence has been presented elsewhere establishing that Zmpste24 possesses a zinc-dependent CAAX endopeptidase activity. In the present study, we confirm this CAAX endopeptidase activity with recombinant, membrane-reconstituted Zmpste24 and show that it can accept a prelamin A farnesylated tetrapeptide as substrate. To monitor the second upstream endoproteolytic cleavage of prelamin A, we expressed a 33 kDa prelamin A C-terminal tail in insect cells. We demonstrate that this purified substrate possesses a C-terminal farnesylated and carboxyl-methylated cysteine and, therefore, constitutes a valid substrate for assaying the second endoproteolytic step in lamin A maturation. With this substrate, we demonstrate that insect cell membranes bearing recombinant Zmpste24 can also catalyse the second upstream endoproteolytic cleavage.

Publisher

Portland Press Ltd.

Subject

Cell Biology,Molecular Biology,Biochemistry

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