4377 Research Article Introduction Quality control mechanisms that operate in the secretory pathway prevent deployment of aberrant proteins to distal compartments. In the endoplasmic reticulum (ER), folding, N- glycosylation and assembly of nascent chains are tightly monitored. Misfolded proteins and orphan subunits of oligomeric proteins, as well as metabolically or developmentally unwanted proteins, are eliminated by the ubiquitin-proteasome system by means of the ER-associated protein degradation (ERAD) pathway (for reviews, see Bonifacino and Weissman, 1998; Ellgaard and Helenius, 2003; Kostova and Wolf, 2003; McCracken and Brodsky, 2003; Sitia and Braakman, 2003; Trombetta and Parodi, 2003; Sayeed and Ng, 2005; Bar-Nun, 2005). Retention of proteins destined for vesicular export is a prerequisite for ERAD; however, retention does not necessarily culminate in degradation. Therefore, it is of interest to determine whether cis-acting motifs that are recognized as ‘retention signals’, preventing protein secretion, overlap with cis-acting motifs that act as ‘degrons’, targeting proteins for degradation. Degrons are defined as sequences or domains that are necessary and sufficient for directing otherwise stable proteins for degradation (Varshavsky, 1991). A degron that targets soluble lumenal proteins for ERAD might play a role in any step along this complex pathway, steps that are not necessarily coupled and schematically entail substrate selection, dislocation to the cytosol, ubiquitylation and degradation by the proteasome. Thus, a degron might constitute the recognition signal for the substrate, it might lead the dislocation process, it might recruit the ubiquitylation machinery and/or provide the platform and Lys residue(s) for ubiquitin conjugation and/or it might target the substrate to the proteasome. The heavy chain of the secretory immunoglobulin M (sIgM), s, is one of the few soluble lumenal ERAD substrates studied in mammalian cells (Amitay et al., 1992; Elkabetz et al., 2003). This protein provides an attractive model to study the interrelations between retention signals and degrons that operate in ERAD. During differentiation of B lymphocytes, the fate of s switches from retention and degradation in B cells to stability and efficient secretion in plasma cells (Amitay et al., 1991; Amitay et al., 1992; Shachar et al., 1992). Moreover, in the same B cell, where s is retained and degraded, the membrane isoform, m, is a stable protein that reaches the cell surface, where it constitutes the B cell receptor. Thus, the intracellular fate of s appears to be mediated by B-cell- specific components and to rely on stp, the C-terminal tailpiece of s that distinguishes it from m. This unique sequence comprises 20 residues that include a penultimate Cys575 and is designated ‘stpCys’. The stpCys motif is a necessary cis-acting retention signal; either its truncation or Cys575 mutation to Ser or Ala hampers retention of s in B cells (Sitia et al., 1990). Moreover, stpCys, unlike stpSer or stpAla, is also sufficient to prevent secretion of IgG2b in B cells or in the non-lymphoid COS-7 cells and to retain the lysosomal protein cathepsin D to a pre-Golgi compartment in COS-7 cells (Sitia et al., 1990; Fra et al., 1993; Isidoro et al., 1996). In addition, the stpCys motif targets the retained Endoplasmic reticulum-associated degradation (ERAD) eliminates aberrant proteins from the secretory pathway. Such proteins are retained in the endoplasmic reticulum and targeted for degradation by the ubiquitin-proteasome system. Cis-acting motifs can function in ERAD as retention signals, preventing vesicular export from the endoplasmic reticulum, or as degrons, targeting proteins for degradation. Here, we show that stp, the C-terminal 20-residue tailpiece of the secretory IgM s heavy chain, functions both as a portable retention signal and as an ERAD degron. Retention of stp fusions of secreted versions of thyroid peroxidase and yellow fluorescent protein in the endoplasmic reticulum requires the presence of the penultimate cysteine of stp. In its role as a portable degron, the stp targets the retained proteins for ERAD but does not serve as an obligatory ubiquitin-conjugation site. Abolishing stp glycosylation accelerates the degradation of both stpCys-fused substrates, yet absence of the N-glycan eliminates the requirement for the penultimate cysteine in the retention and degradation of the unglycosylated yellow fluorescent protein. Hence, the dual role played by the stpCys motif as a retention signal and as a degron can be attributed to distinct elements within this sequence. Key words: ERAD, Retention signals, Degrons, Proteasome Summary Distinguishing between retention signals and degrons acting in ERAD Ilana Shapira 1 , Dana Charuvi 1, * , Yechiel Elkabetz 1,‡ , Koret Hirschberg 2 and Shoshana Bar-Nun 1,§ 1 Department of Biochemistry, George S. Wise Faculty of Life Sciences and 2 Department of Pathology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel *Present address: The Robert H Smith Institute of Plant Sciences and Genetics in Agriculture, The Hebrew University of Jerusalem, Rehovot, Israel ‡ Present address: Laboratory of Stem Cell and Tumor Biology, Division of Neurosurgery and Developmental Biology Program, Sloan-Kettering Institute, New York, NY, USA § Author for correspondence (e-mail: shoshbn@tauex.tau.ac.il) Accepted 3 October 2007 Journal of Cell Science 120, 4377-4387 Published by The Company of Biologists 2007 doi:10.1242/jcs.011247 Journal of Cell Science