Abstract
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 the cis-Golgi cisternae in pancreatic acinar cells (Lahtinen, U., Dahllöf, B., and Saraste, J. (1992) J. Cell Sci. 103, 321-333). Here we report 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 back to the endoplasmic reticulum. The rat p58 sequence was found to be 89% identical with those of ERGIC-53 and MR60, two previously identified human membrane proteins. Strong homology with the frog sequence was also observed indicating high evolutionary conservation. Overexpression 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 using antibodies raised against a lumenal peptide stained the total cellular 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 intermediates is hidden, but becomes exposed when the protein reaches the Golgi complex.
Highlights
The endomembranes participating in the early stages of protein maturation and transport consist of the endoplasmic reticulum (ER),1 the cis-Golgi cisternae and tubulo-vesicular elements at the interface between these organelles (Palade, 1975)
We report here that p58 is the rat homolog of the human ERGIC-53/MR60 protein (Schindler et al, 1993; Arar et al, 1995), which has been suggested to function as a membrane bound lectin in the early secretory pathway (Fiedler and Simons, 1994; Arar et al, 1995)
During the last several years p58 (Saraste et al, 1987; Saraste and Svensson, 1991; Lahtinen et al, 1992) and p53 (Schweitzer et al, 1988, 1990; renamed ERGIC-53, Schindler et al (1993)), two membrane proteins identified in rat and human cells, respectively, have been widely used as markers for the intermediate compartment at the ER-Golgi interface
Summary
The endomembranes participating in the early stages of protein maturation and transport consist of the endoplasmic reticulum (ER),1 the cis-Golgi cisternae and tubulo-vesicular elements at the interface between these organelles (Palade, 1975). We report 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.
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