Gain- and loss-of-function analyses reveal an important role for CREG1 in both macropinocytosis and clathrin-dependent endocytosis. antigen 9; EIPA: 5-(N-ethyl-N-isopropyl)amiloride; ER: endoplasmic reticulum; GFP: green fluorescent protein; HEXA: hexosaminidase subunit alpha; IGF2R: insulin like growth factor 2 receptor; LAMP1: lysosomal associated membrane protein 1; M6PR: mannose-6-phosphate receptor, cation dependent; MAPK1/ERK2: mitogen-activated protein kinase 1; MTORC1: mechanistic target of rapamycin kinase complex 1; PDIA2: protein disulfide isomerase family A member 2; SQSTM1/p62: sequestosome 1; TF: transferrin; TFEB: transcription factor EB KEYWORDS: Autophagy, endocytosis, gene targeting, hepatocytes, immunofluorescence Introduction (Cellular repressor of E1A stimulated genes) was cloned in yeast two-hybrid screening of a cDNA library [1]. It was initially described as a BCX 1470 transcription repressor that antagonized the transcription and cellular transformation induced by the adenovirus E1A oncoprotein. Later CREG was found to be a glycoprotein that could be secreted into cell culture media [2]. Two paralogs have been identified in the CREG family, CREG1 and CREG2 [3]. mRNA is ubiquitously expressed while was only detected in the brain [2]. CREG1 has a signal peptide (amino acids 1C31 in human and mice, 1C23 in CREG-GFP was also detected in lysosomes in S2 cells [4]. Recently, CREG1 was shown to partially colocalize with mitochondrial UCP1 (uncoupling protein 1) [10]. In addition, CREG1 could be detected in conditioned media, serum, and urine [2,11C13]. Therefore, it has been suggested as a secretory protein. Morphological evidence for CREG1 localization in lysosomes is supported by mass spectrometric analysis of lysosomal proteins. Carbohydrate chains in a majority of soluble lysosomal proteins consist of the mannose 6-phosphate (M6P) tag, which is recognized by M6P receptors [14]. There are two such receptors, the 46-kD cation-dependent M6PR (mannose-6-phosphate receptor, cation dependent) and the 300-kD cation-independent IGF2R (insulin like growth factor 2 receptor). CREG1 binds to IGF2R in far-western blot analysis [15]. Journet et al. used IGF2R affinity purification and mass spectrometry to identify CREG1 from conditioned media of human monocytic U937 cells, which were induced to BCX 1470 secrete lysosomal proteins by NH4Cl [16,17]. Proteomic analysis of lysosomes isolated from the mouse liver suggested CREG1 to Rabbit polyclonal to Shc.Shc1 IS an adaptor protein containing a SH2 domain and a PID domain within a PH domain-like fold.Three isoforms(p66, p52 and p46), produced by alternative initiation, variously regulate growth factor signaling, oncogenesis and apoptosis. be a lysosomal protein [18]. Macroautophagy/autophagy is an evolutionarily conserved lysosome pathway that degrades cytoplasmic components and organelles [19]. It is critical for embryonic development and normal physiology, and its dysregulation is associated with various diseases, including neurodegeneration, diabetes, and cancer. However, the role of CREG1 in autophagy is poorly understood. In this study, we validated CREG1 antibodies for immunostaining and demonstrate that CREG1 is localized to the endosomal-lysosomal compartment. Gain- and loss-of-function analyses reveal that CREG1 promotes lysosomal biogenesis, acidification, and degradation, thereby accelerating autophagic flux. These effects are likely mediated by enhanced endocytic trafficking. Results CREG1 is an endosomal-lysosomal protein highly expressed in the liver, lung, fat, and immune organs/cells mRNA was shown to be ubiquitously expressed in adult mouse tissues with the highest expression level detected in the liver [2]. To determine CREG1 protein expression in mouse tissues, we performed immunoblot analysis on tissue lysates as well as leukocytes. The CREG1 protein was highly expressed in the spleen, liver, lung, and white adipose tissues as well as leukocytes and bone marrow (Figure 1A). In these tissues, CREG1 was mainly detected as a ~?22 kDa band. However, in the heart the major band is ~17 kDa. Previous studies showed that CREG1 was localized to the ER, lysosome, or mitochondria using house made or uncharacterized, commercially available antibodies [2,5,10]. To screen for CREG1 antibodies suitable BCX 1470 for immunostaining, we employed LO2 human hepatocytes, which express relatively low level of endogenous CREG1 and are easy to transfect using cationic lipid transfection reagents. LO2 cells have been re-cloned in our laboratory based on a cobblestone epithelial morphology and higher expression levels of mRNAs for (albumin), (coagulation.