Characterization of p58 and rab1p, two proteins operating at the interface of the endoplasmic reticulum and the Golgi complex
Author: Lahtinen, Ulla
Date: 1998-04-17
Location: Auditoriet, vid Institutionen för Cell- och molekylärbiologi
Time: 10.00
Department: Inst för cell- och molekylärbiologi / Dept of Cell and Molecular Biology
Abstract
Endomembranes participating in the early stages in exocytosis consist of the endoplasmic reticulum (ER), the Golgi complex, and vesicular transport intermediates at their interface. These vesicular transport intermediates were first visualized by following the synchronized transport of a viral membrane protein from the ER to the Golgi complex, which occurs at slower rates at reduced temperatures causing an accumulation of transport vesicles. Identification of a 58 kDa membrane protein, p58, in rat pancreatic cells, which localized to the cis-Golgi cisternae and associated tubules and vesicles, provided the first resident marker protein to study the morphology of the ER-Golgi boundary and transport dynamics between these organelles by immunocytochemistry. Co-localization of p58 with newly synthesized viral membrane proteins indicated a role of these p58-positive vesicular structures in biosynthetic membrane transport.
An immunocytochemical analysis revealed the presence of p58 in multiple cellular compartments. In addition to the cis-Golgi cisterna, p58 was detected in small Golgi vesicles and buds, in larger pleomorphic vesicular elements found both throughout the cell cytoplasm and associated with smooth ER, and at lower levels also in rough ER cisternae. Rab1p, a small GTP-binding protein involved in ER to Golgi transport, was detected on one or two cis-most Golgi cisternae, in tubulo-vesicular membranes in close vicinity for the cis-Golgi region and also widely distributed in the cell, but absent from the rough ER. A local labeling between closely opposed membranes could also be seen. This indicates that rab1p is recruited into post-ER, pre Golgi membranes and may function in targeting or fusion of pre- and cis-Golgi membranes.
p58 was shown to be a major disulfide-linked homodimeric and -hexameric integral membrane protein in microsomes. It was purified into homogeneity by two dimensional gel electrophoresis to obtain peptide sequences. The isolated rat p58 cDNA encoded a 517 amino acid protein, having a putative signal sequence, a transmembrane domain close to the C-terminus and a short cytoplasmic tail containing the KKXX-signal known to mediate recycling of a number of proteins by retrieval from the Golgi complex back to the ER. Sequence comparison showed 89% identity to the human intermediate compartment ERGIC-53 protein and the human mannose-binding protein, MR60. Rat p58 was 71% identical to the frog sequence and 36% identical to a worm sequence indicating conservation during evolution.
The lumenal domains of p58 and VIP36, a membrane protein in exocytic carrier vesicles, were found to be related sharing 30% identity. Since VIP36 has been shown by others to be structurally related to plant leguminous lectins, having conserved sugar and metal binding residues, a new family of animal lectins operating in different stages along the secretory pathway was proposed. Isolation of a human mannose binding protein, MR60, and its identity to ERGIC-53, the human homologue of p58, strengthened the lectin hypothesis. p58 might function as a receptor in concentrating and packaging newly synthesized glycoproteins into transport vesicles, retrieving misfolded glycoproteins back to the ER, or maintaining the topology or localization of lipid-linked high-mannose oligosaccharides.
An immunocytochemical analysis revealed the presence of p58 in multiple cellular compartments. In addition to the cis-Golgi cisterna, p58 was detected in small Golgi vesicles and buds, in larger pleomorphic vesicular elements found both throughout the cell cytoplasm and associated with smooth ER, and at lower levels also in rough ER cisternae. Rab1p, a small GTP-binding protein involved in ER to Golgi transport, was detected on one or two cis-most Golgi cisternae, in tubulo-vesicular membranes in close vicinity for the cis-Golgi region and also widely distributed in the cell, but absent from the rough ER. A local labeling between closely opposed membranes could also be seen. This indicates that rab1p is recruited into post-ER, pre Golgi membranes and may function in targeting or fusion of pre- and cis-Golgi membranes.
p58 was shown to be a major disulfide-linked homodimeric and -hexameric integral membrane protein in microsomes. It was purified into homogeneity by two dimensional gel electrophoresis to obtain peptide sequences. The isolated rat p58 cDNA encoded a 517 amino acid protein, having a putative signal sequence, a transmembrane domain close to the C-terminus and a short cytoplasmic tail containing the KKXX-signal known to mediate recycling of a number of proteins by retrieval from the Golgi complex back to the ER. Sequence comparison showed 89% identity to the human intermediate compartment ERGIC-53 protein and the human mannose-binding protein, MR60. Rat p58 was 71% identical to the frog sequence and 36% identical to a worm sequence indicating conservation during evolution.
The lumenal domains of p58 and VIP36, a membrane protein in exocytic carrier vesicles, were found to be related sharing 30% identity. Since VIP36 has been shown by others to be structurally related to plant leguminous lectins, having conserved sugar and metal binding residues, a new family of animal lectins operating in different stages along the secretory pathway was proposed. Isolation of a human mannose binding protein, MR60, and its identity to ERGIC-53, the human homologue of p58, strengthened the lectin hypothesis. p58 might function as a receptor in concentrating and packaging newly synthesized glycoproteins into transport vesicles, retrieving misfolded glycoproteins back to the ER, or maintaining the topology or localization of lipid-linked high-mannose oligosaccharides.
Issue date: 1998-03-27
Publication year: 1998
ISBN: 91-628-2927-0
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