Carboxyl-terminal Targeting and Novel Post-translational Processing of JAW1, a Lymphoid Protein of the Endoplasmic Reticulum

Timothy W. Behrens, Grainne M. Kearns, James J. Rivard, Harris D. Bernstein, Jonathan W. Yewdell, Louis M. Staudt
1996 Journal of Biological Chemistry  
Jaw1 is a lymphoid-restricted protein localized to the cytoplasmic face of the endoplasmic reticulum (ER) and is a member of a recently recognized class of integral membrane proteins that contain carboxyl-terminal membrane anchors. The carboxyl-terminal 71 amino acids of the Jaw1 protein, which contain a hydrophobic membrane spanning region, are sufficient to target a heterologous protein to the ER. By discontinuous sucrose gradient ultracentrifugation, differential sedimentation was noted for
more » ... he four major Jaw1 protein isoforms, with two of the forms predominantly soluble and two microsome-bound. Pulse-chase immunoprecipitations suggest a post-translational modification of two major isoforms of the protein resulting in an increase in mobility on SDS-polyacrylamide gel electrophoresis. In vitro translation studies are compatible with a posttranslational processing event that results in cleavage of a short 36 amino acid lumenal domain. These findings define a carboxyl-terminal domain of the Jaw1 protein that is both necessary and sufficient for ER localization. In addition, the processing of the small lumenal domain of Jaw1 represents a novel post-translational protein modification performed by the endoplasmic reticulum. The insertion of type I membrane proteins (N exo C cyt ) and all secreted proteins into the endoplasmic reticulum generally occurs co-translationally and requires the participation of signal recognition particle (SRP) 1 (1, 2). The 54-kDa subunit of SRP binds to the amino-terminal signal sequence of nascent protein chains and delivers the nascent protein-ribosome complex to the ER membrane where docking and translocation of the nascent chain into the ER lumen commences (3, 4). After cleavage of the signal peptide in the lumen of the ER by signal peptidase, translocation of the polypeptide proceeds (reviewed Most type II proteins (N cyt C exo ) possess an uncleaved hydrophobic domain near the amino terminus that serves as both a signal sequence and an anchor domain (6). Following targeting of a type II nascent protein chain and ribosome to the ER membrane by SRP, the carboxyl terminus is translocated across the ER membrane while the amino terminus remains cytosolic. The charged amino acids that flank hydrophobic transmembrane (TM) domains are thought to play an important role in determining the ultimate orientation of the protein in the membrane. According to the "positive inside" rule of von Heijne, the flanking region with the greatest net positive charge is generally oriented facing the cytosol (7). A survey of a large number of integral membrane proteins confirmed this prediction, showing that the difference in charges of the 15 amino acids on either side of the first internal signal-anchor determines the topology of the mature protein (8). By mutating key charged residues that flank the TM domain of a type II viral protein, topology could be inverted to a type I orientation (9). The mechanism by which these charge differences are sensed at the ER membrane is not known (10). Recently, a class of integral membrane proteins with carboxyl-terminal membrane anchors (CTMAs) was identified (11). This class includes a large number of proteins that are important for the targeting and fusion of intracellular vesicles (v-and t-SNARE (soluble NSF attachment protein receptor) proteins) (12), membrane-bound protein tyrosine phosphatases, cytochromes, and others (see Ref. 11). Proteins of this class are characterized by the presence of a carboxyl-terminal membrane spanning domain without an amino-terminal signal sequence and are oriented facing the cytosol. The mechanism by which CTMA proteins target to and insert in membranes has not been well studied. In a recent study, we reported the cloning and initial characterization of a lymphoid-restricted protein called Jaw1 (13). Jaw1 has a coiled-coil domain in the middle-third of the protein and a carboxyl-terminal membrane anchor with a conserved 36-amino acid lumenal tail. Expression of Jaw1 mRNA and protein is developmentally regulated in both the B and T cell lineages with highest expression in pre-T, pre-B, and mature B cells. Several lines of evidence suggest that Jaw1 protein is expressed exclusively in the endoplasmic reticulum. The protein co-localized with the ER marker BiP in lymphocytes by indirect immunofluorescence and confocal microscopy. When transfected into Hela cells, which lack the protein, Jaw1 targeted to the ER as demonstrated by co-localization with the ER lumenal protein, PDI. Furthermore, in an in vitro translation system the Jaw1 protein inserts into pancreatic microsomes and behaves as an integral membrane protein. Thus, these
doi:10.1074/jbc.271.38.23528 pmid:8798562 fatcat:ngosdxgssbbnvkh4mdqvbchd54