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Lamp2s and Chaperone-Mediated Autophagy 4443 of Trichloroacetic Acid to a final Concentration of 10% Journal of Cell Science 113, 4441-4450 (2000) 4441 Printed in Great Britain © The Company of Biologists Limited 2000 JCS1804 Unique properties of lamp2a compared to other lamp2 isoforms A. M. Cuervo* and J. F. Dice Department of Physiology, Tufts University School of Medicine, Boston, MA, USA *Author for correspondence (e-mail: [email protected]) Accepted 28 September; published on WWW 16 November 2000 SUMMARY Lamp2a acts as a receptor in the lysosomal membrane for other lamp2s. Four positively-charged amino acids substrate proteins of chaperone-mediated autophagy. uniquely present in the cytosolic tail of lamp2a are required Using antibodies specific for the cytosolic tail of lamp2a and for the binding of substrate proteins. Lamp2a also others recognizing all lamp2 isoforms, we found that in rat distributes to an unique subpopulation of perinuclear liver lamp2a represents 25% of lamp2s in the lysosome. We lysosomes in cultured fibroblasts in response to serum show that lamp2a levels in the lysosomal membrane in withdrawal, and lamp2a, more than other lamp2s, tends to rat liver and fibroblasts in culture directly correlate with multimerize. These characteristics may be important for rates of chaperone-mediated autophagy in a variety lamp2a to act as a receptor for chaperone-mediated of physiological and pathological conditions. The autophagy. concentration of other lamp2s in the lysosomal membrane show no correlation under the same conditions. Key words: Lysosome, Protein degradation, Membrane receptor, Furthermore, substrate proteins bind to lamp2a but not to Chaperone, Rat liver INTRODUCTION Part of this matrix form of lamp2 reversibly aggregates with other lysosomal enzymes in a pH-dependent manner (Jadot et Lysosome-associated membrane proteins (lamps) are a al., 1997). The origin of the lumenal form of lamp2a is still group of lysosomal proteins with very similar structural unclear, but a direct deinsertion from the lysosomal membrane characteristics but of mostly unknown function (Fukuda, 1991; after a conformational change, as well as a release by Peters and von Figura, 1994). All of the lamps are type I proteolytic cleavage of the short transmembrane and cytosolic integral glycosylated proteins with a large lumenal domain, a tail have been proposed (Jadot et al., 1996). That kind of single transmembrane region of about 20 amino acids, and a deinsertion has been described for other type I membrane short (10-12 amino acid) carboxyl terminus tail at the cytosolic proteins (Nishiyama et al., 1999), and cleavage has been side of the lysosomal membrane (Akasaki and Tsuji, 1998). described for other lysosomal membrane proteins such as acid Lamps are mainly localized to lysosomes but can also be phosphatase (Gottschalk et al., 1989) and lamp1 (Meikle et al., detected in lower amounts in endosomes and at the plasma 1999). The high level of glycosylation of lamp2, in which the membrane (Furuno et al., 1989; Akasaki et al., 1993). Two protein core accounts for only 40 kDa of the final glycosylated different classes of lamps, lamp1 and lamp2, encoded by two product of 96 kDa, and the small size of the transmembrane different but evolutionarily-related genes have been described. and cytosolic tail, make it difficult to identify the matrix The lamp2 gene undergoes alternative splicing resulting in at forms of lamp2 as intact or truncated using conventional least three different mRNAs encoding different isoforms of electrophoretic methods. lamp2 (Gough et al., 1995; Hatem et al., 1995; Konecki et al., The function of most lamps remains unclear. They have been 1995). The three lamp2 isoforms (a, b and c) identified so far hypothesized to play a role in protecting the lysosomal show high amino acid sequence identity in their lumenal membrane from its associated hydrolases (Fukuda, 1991). region, but different transmembrane and cytosolic regions However, it has been shown recently that the complete (Gough et al., 1995). The lamp2 splice variants are expressed elimination of lamp1 that constitutes almost 40% of the at different levels in different tissues (Konecki et al., 1995; lysosomal membrane protein does not modify lysosomal Furuta et al., 1999) and have different distributions between the stability (Andrejewski et al., 1999). A role for lamp2 in cell- plasma membrane and lysosomes (Gough and Fambrough, cell or cell-extracellular matrix adhesion has been proposed 1997). Lamp2 is concentrated in tissues undergoing apoptosis (Lippincott-Schwartz and Fambrough, 1986; Carlsson et al., during development, and the expression pattern for each lamp2 1988; Saitoh et al., 1992; Licheter-Konecki et al., 1999). A role isoform becomes more tissue and cell-type specific as for lamp2 in maturation of autophagic vacuoles has also differentiation progresses (Licheter-Konecki et al., 1999). been proposed (Tanaka et al., 2000). By analogy with other Lamp2s are present in the lysosomal lumen as well as the alternatively spliced proteins (Ravetch and Perussia, 1989), the lysosomal membrane (Jadot et al., 1996; Jadot et al., 1997). tissue-dependent expression of the different forms of lamp2 4442 A. M. Cuervo and J. F. Dice (Konecki et al., 1995) suggests that they might have different of serum, plates were extensively washed with Hanks’ balanced salts cellular functions. solution (Life Technologies, Gaithersburg, MD) and medium without We identified the lamp type 2a (lamp2a) at the lysosomal serum was added. membrane as a receptor for substrates of chaperone-mediated Chemicals autophagy (Cuervo and Dice, 1996). In this pathway, specific cytosolic proteins are directly transported through the Sources of chemicals and antibodies were as described previously (Terlecky and Dice, 1993; Cuervo et al., 1994; Cuervo et al., 1995; lysosomal membrane into the lysosomal matrix where they are Cuervo and Dice, 1996). The antibody against the cytosolic tail of rat degraded (Cuervo and Dice, 1998; Dice, 2000). Substrate lamp2a was raised in our laboratory (Cuervo and Dice, 1996). The proteins bind first to a constitutively expressed heat shock monoclonal antibodies against the lumenal side of rat lamp2a, rat protein of 73 kDa (hsc73) in the cytosol that targets them to lamp1, human influenza hemaglutinin protein (HA) and cathepsin A lysosomes (Chiang et al., 1989). A second chaperone located were gifts from Dr Michael Jadot (Facultes Universitaires Notre- in the lumen of the lysosomes, the lysosomal hsc73, is required Dame de la Paix, Namur, Belgium), Dr Ira Mellman (Yale University for the complete transport of substrate proteins into lysosomes School of Medicine, New Heaven, CT), Dr Anjana Rao (Department (Agarraberes et al., 1997; Cuervo et al., 1997). We of Pathology, Harvard Medical School, Boston, MA), and Dr demonstrated that substrate proteins bind to the cytosolic tail Alessandra D’Azzo, respectively. The monoclonal antibodies against of lamp2a at the lysosomal membrane before their transport the matrix region of human, mouse, and hamster lamp2s, and human lamp1 were obtained from the Developmental Studies Hybridoma and degradation in the lysosomal matrix (Cuervo and Dice, Bank (Iowa City, IA). Aminolink and Sulfolink gels and 1996). Inhibition of that binding with specific antibodies crosslinking agents were from Pierce (Rockford, IL). against the cytosolic tail of lamp2a or by competition with a synthetic peptide of the same amino acid sequence as the tail, Isolation of subcellular fractions results in blocking of substrate uptake and degradation in Rat liver lysosomes were isolated from a light mitochondrial- lysosomes (Cuervo and Dice, 1996). Interestingly, the lysosomal fraction in a discontinuous metrizamide density gradient overexpression of only lamp2a in cultured cells increased the (Wattiaux et al., 1978) by the shorter method reported previously rates of chaperone-mediated autophagy, suggesting that (Aniento et al., 1993). After isolation lysosomes were resuspended binding of the substrates to lamp2a at the lysosomal membrane in MOPS buffer (0.3 M sucrose/10 mM 3-(N-morpholino) might be a rate-limiting step in this pathway (Cuervo and Dice, propanesulfonic acid (MOPS), pH 7.2). In some experiments, two separate lysosomal fractions with different levels of chaperone- 1996). mediated autophagy were isolated as described (Cuervo et al., 1997). In the present study, using an antibody specific for lamp2a Contamination of the lysosomal fraction with mitochondria (based and another that recognizes all lamp2s we compare on the activity of ornithine transcarbamoylase and succinate concentrations, biochemical properties, subcellular location, dehydrogenase) or cytosol (based on the activity of lactate and lysosomal distribution of lamp2s in rat liver and fibroblasts dehydrogenase and GAPDH) could account for less than 0.5% of the in culture, the two systems in which chaperone-mediated lysosomal fraction. Lysosomes from cultured cells were isolated as autophagy has been well-characterized. We demonstrate that described (Storrie and Madden, 1990). Integrity of the lysosomal binding of substrate proteins to lamp2a is a rate-limiting membrane after isolation was measured by β-hexosaminidase latency step in chaperone-mediated autophagy under a variety of as previously described (Terlecky and Dice, 1993). Only preparations physiological and pathological conditions, while the other with more than 95% intact lysosomes were used. Lysosomal matrices and membranes were obtained as described by Oshumi (Ohsumi et lysosomal forms of lamp2a do not affect chaperone-mediated al.,
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