MOLECULAR-CLONING AND EXPRESSION OF A 58-KDA CIS-GOLGI AND INTERMEDIATE COMPARTMENT PROTEIN

An abundant 58-kDa (p58) homodimeric and hexameric microsomal membrane protein has been biochemically characterized and localized to tubulo- vesicular elements at the endoplasmic reticulum-Golgi interface and th e cis-Golgi cisternae in pancreatic acinar cells (Lahtinen, U., Dahllo f, B., and Saraste, J. (1992) J. Cell Sci. 103, 321-333). Here we repo rt the purification of p58 by two-dimensional gel electrophoresis, and the cloning and sequencing of the rat and part of the Xenopus laevis cDNAs. The rat cDNA encodes a 517-amino acid protein having a putative signal sequence, a transmembrane domain close to the C terminus and a short cytoplasmic tail. The C-terminal tail contains a double-lysine motif (KKFF), known to mediate retrieval of proteins from the Golgi ba ck to the endoplasmic reticulum. The rat p58 sequence was found to be 89% identical with those of ERGIC-53 and MR60, two previously identifi ed human membrane proteins. Strong homology with the frog sequence was also observed indicating high evolutionary conservation. Overexpressi on of c-Myc-tagged p58 resulted in accumulation of the protein both in the endoplasmic reticulum and in an apparently enlarged Golgi complex , as well as its leakage to the plasma membrane. Immunolocalization us ing antibodies raised against a lumenal peptide stained the total cell ular pool of p58, while anti-tail peptide antibodies detected p58 only in a restricted Golgi region. This suggests that the C-terminal tail of p58 located in the endoplasmic reticulum and transport intermediate s is hidden, but becomes exposed when the protein reaches the Golgi co mplex.