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The Efficiency of Protein Compartmentalization Into The Molecular Biology of the Cell Vol. 16, 279–291, January 2005 The Efficiency of Protein Compartmentalization into the Secretory Pathway□D Corinna G. Levine,* Devarati Mitra,* Ajay Sharma,* Carolyn L. Smith,† and Ramanujan S. Hegde*‡ *Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892; and †Light Imaging Facility, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892 Submitted June 22, 2004; Revised September 21, 2004; Accepted October 5, 2004 Monitoring Editor: Reid Gilmore Numerous proteins targeted for the secretory pathway are increasingly implicated in functional or pathological roles at alternative cellular destinations. The parameters that allow secretory or membrane proteins to reside in intracellular locales outside the secretory pathway remain largely unexplored. In this study, we have used an extremely sensitive and quantitative assay to measure the in vivo efficiency of signal sequence-mediated protein segregation into the secretory pathway. Our findings reveal that segregation efficiency varies tremendously among signals, ranging from >95 to <60%. The nonsegregated fraction is generated by a combination of mechanisms that includes inefficient signal-mediated translocation into the endoplasmic reticulum and leaky ribosomal scanning. The segregation efficiency of some, but not other signal sequences, could be influenced in cis by residues in the mature domain or in trans by yet unidentified cellular factors. These findings imply that protein compartmentalization can be modulated in a substrate-specific manner to generate biologically significant quantities of cytosolically available secretory and membrane proteins. INTRODUCTION regulates p21 translation (Iakova et al., 2004). In each of these instances, both functional and physical interactions with the In mammalian cells, an estimated one-fifth to one-third of all respective cytosolic components have been demonstrated. proteins reside in or transit through the secretory pathway. Similarly, a different ER resident protein, the ␤ subunit of The decisive step in segregating these proteins from the glucosidase II (Trombetta et al., 1996), was originally discov- cytosol is their cotranslational targeting to and translocation ered (and given the name 80K-H) as a target of protein across the endoplasmic reticulum (ER) membrane (reviewed kinase C (Sakai et al., 1989; Hirai and Shimizu, 1990). More by Walter and Johnson, 1994; Rapoport et al., 1996; Johnson recently, it has been demonstrated to interact with the cyto- and van Waes, 1999). Both targeting and initiation of trans- solic adaptor protein Grb3 during signal transduction (Goh location are dependent on a signal sequence, often at the N et al., 1996; Kanai et al., 1997), shuttle between the nucleus terminus, encoded in secretory and membrane proteins. Al- and cytosol (Forough et al., 2003), and modulate calcium though the primary sequence of signals varies broadly (von homeostasis by binding to the cytosolic tail of the TRP5 Heijne, 1985), they contain shared features (such as hydro- channel (Gkika et al., 2004). Other secretory pathway pro- phobicity) that allow their common recognition by a ubiq- teins with alternative localizations include mitochondrial uitous machinery for ER translocation. Thus, proteins con- transforming growth factor-␤ (Heine et al., 1991), mitochon- taining signal sequences are generally considered to have drial Slit3 (Little et al., 2001), mitochondrial cytochrome p450 their intended functions at destinations accessed via the (Addya et al., 1997; Anandatheerthavarada et al., 1999), cy- secretory pathway. tosolic IL15 (Tagaya et al., 1997; Kurys et al., 2000), nucleo- Increasingly, however, many signal-containing proteins cytoplasmic cathepsin L (Goulet et al., 2004), and nuclear have been implicated in functional, pathological, or yet un- HBV precore protein (Garcia et al., 1988; Ou et al., 1989). known roles at sites outside the secretory pathway. For Alternative localizations of secretory pathway proteins example, the ER lumenal chaperone calreticulin (CRT) was also are associated with various disease states. Although identified completely independently as a cytosolic integrin- some observations of unusual localizations simply correlate binding protein (Leung-Hagesteijn et al., 1994; Coppolino et with a disease state (Ramsamooj et al., 1995; Yoon et al., 2000; al., 1995, 1997), a nuclear export factor (Holaska et al., 2001), Brunagel et al., 2003), other examples have been mechanis- a regulator of steroid hormone receptor activity (Burns et al., tically implicated in contributing to pathogenesis. For exam- 1994; Dedhar et al., 1994), and an mRNA binding protein that ple, the secretory protein apolipoprotein E (apoE) has been suggested to interact with the neurofilament Tau in the cytosol to induce neurofibrillary tangles (Huang et al., 2001; Article published online ahead of print. Mol. Biol. Cell 10.1091/ Ljungberg et al., 2002; Harris et al., 2003; Brecht et al., 2004), mbc.E04–06–0508. Article and publication date are available at a common feature of Alzheimer’s disease. That only the www.molbiolcell.org/cgi/doi/10.1091/mbc.E04–06–0508. □ disease-associated polymorphic form (apoE4) but not pro- D The online version of this article contains supplemental material tective form (apoE3) had these effects further supports such at MBC Online (http://www.molbiolcell.org). a role in the cytosol. Similarly, another secretory pathway ‡ Corresponding author. E-mail address: [email protected]. protein involved in Alzheimer’s disease, APP, was shown to © 2004 by The American Society for Cell Biology 279 C. G. Levine et al. be capable of translocating partially into mitochondria and nonsecretory pathway compartment? If so, what are the inducing apoptosis (Anandatheerthavarada et al., 2003). Fur- mechanisms and parameters that influence how much is thermore, a pathogenic fragment of APP, a-␤, seems to localized to the alternative compartment? Is inefficiency of interact with and inhibit a key mitochondrial enzyme (Lust- compartmentalization an inherent property of any given bader et al., 2004). Cytosolically localized prion protein (PrP) substrate or can it be selectively modulated? The answers to also has been suggested by recent studies to be harmful and these questions may help provide a common framework for associated with neurodegeneration (Ma et al., 2002). Yet understanding the otherwise disparate observations of un- other studies have observed PrP in the cytosol under non- expected or aberrant locales of many physiologically impor- pathogenic conditions (Mironov et al., 2003), where it may tant proteins. interact with and protect against Bax-mediated cell death (Bounhar et al., 2001; Roucou et al., 2003). MATERIALS AND METHODS For most of these signal sequence-containing proteins, the mechanisms or extent to which they arrive at their alterna- DNA Constructs tive localizations remains largely a matter of speculation (for The coding region for the heterologous Gal4-nulcear factor-␬B (NF-␬B) tran- a notable exception, see Goulet et al., 2004). It is therefore scription factor (TF) was amplified by polymerase chain reaction (PCR) from important to know, with a high degree of sensitivity and plasmid pBD-NF-␬B (Stratagene, La Jolla, CA) and subcloned in frame into a calreticulin expression construct at the unique NotI site just preceding the precision, the efficiency by which signal sequence-contain- coding region for the C-terminal KDEL ER retention sequence. The region ing substrates are segregated to the ER in vivo. Although coding for TF, together with the KDEL sequence, was PCR amplified and small differences in efficiency (for example 95 vs. 99%) subcloned into a blunted PstI site and a downstream XbaI site of cassettes would have little or no consequences for the secretory path- containing different signal sequences. Such signal sequence cassettes have been described previously (Kim et al., 2002). Changes to the region between way form of the substrate, it would have substantial impli- the signal and mature domain (such as the inclusion of 10 amino acids from cations for the alternative form (which in this example, the respective mature domain, or various mutations as described in the text) would differ by a factor of 5). Such seemingly small propor- were encoded within the oligonucleotides used for PCR amplification of TF. tions (e.g., even 1% or less) of abundant secretory pathway Some point mutants were generated by site-directed mutagenesis by using the QuikChange method (Stratagene). The vesicular stomatitis virus glycop- proteins (such as CRT, 80K-H, or PrP, among others) would rotein (VSVG)-green fluorescent protein (GFP) construct comprising the ts045 still easily be within the range of physiologically relevant temperature-sensitive VSVG with a C-terminally appended enhanced green protein abundances. This is particularly true when the pu- fluorescent protein (EGFP) has been described previously (Presley et al., 1997). tative alternative form has proposed functions, such as tran- The mature domain of this plasmid was PCR amplified and subcloned down- stream of the Prl or PrP signal sequences by using the cassettes described scriptional regulation (Burns et al., 1994; Dedhar et al., 1994) above. A similar strategy was used to replace the PrP signal sequence with or signal transduction (Goh et al., 1996; Kanai et al., 1997) that those from Osteopontin (Opn) or
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