Microsomal aldehyde dehydrogenase is localized to the endoplasmic reticulum via its carboxyl-terminal 35 amino acids.

Abstract

Abstract. Rat microsomal aldehyde dehydrogenase (msALDH) has no amino-terminal signal sequence, but instead it has a characteristic hydrophobic domain at the carboxyl terminus (Miyauehi, K., R. Masaki, S. Taketani, A. Yamamoto, A. Akayama, and Y. Tashiro. 1991. J. Biol. Chem. 266:19536-19542). This membrane-bound enzyme is a useful model protein for studying posttranslational localization to its final destination. When expressed from eDNA in COS-1 cells, wild-type msALDH is localized exclusively in the well-developed ER. The removal of the hydrophobic domain results in the cytosolic localization of truncated proteins, thus suggesting that the portion is responsible for membrane anchoring. The last 35 amino acids of msALDH, including the hydrophobic domain, are sufficient for targeting of E. coli ~5-galactosidase to the ER membrane. Further studies using chloramphenicol acetyltransferase fusion proteins suggest that two hydrophilie sequences on either side of the hydrophobic domain play an important role in ER targeting.great deal of attention has been paid to resolve the mechanism for sorting and targeting of newly synthesized proteins to their final destinations, and this is one of the fundamental problemsin cell biology. Newly synthesized proteins are destined to follow two distinct routes, depending on the presence or absence of a signal sequence at their amino termini (37). The signal recognition particle (SRP) t in the cytosol recognizes and binds to a signal sequence of nascent peptides. The resulting SRP-ribosome-nascent peptides complexes are targeted to the ER membrane through their interactions with the docking protein complex (35,36). After translocation across the ER membrane, secretory and plasma membrane proteins follow a common pathway from the ER, through the Golgi complex, to the cell surface by default with bulk flow of lipids (33). Resident proteins in the central vacuolar system are localized and retained in their final destinations with the aid of either specific targeting (18) or retention signals (25)(26)(27)44).On the other hand, proteins without a signal sequence at their amino termini are synthesized on free polysomes and

The Role of the 3-Hydroxy 3-Methylglutaryl Coenzyme A Reductase Cytosolic Domain in Karmellae Biogenesis

“…Cheng and coworkers (1999) did not analyze the ability of their fusion proteins to induce crystalloid ER. However, studies of microsomal aldehyde dehydrogenase, which induces crystalloid ER similar to that induced by HMG-CoA reductase, also indicate the importance of interactions via the carboxyl termini for induction of membrane assembly (Masaki et al, 1994Gong et al, 1996;Yamamoto et al, 1996). Cheng et al (1999) propose that dimerization via the HMG-CoA reductase catalytic domains serves to promote or to stabilize interactions between the attached membrane domains.…”

The GPI Transamidase Complex ofSaccharomyces cerevisiaeContains Gaa1p, Gpi8p, and Gpi16p

“…In the situation of overexpression, the ER folding machinery may get overwhelmed, and incompletely folded proteins may accumulate and aggregate in a nonspecific way through hydrophobic interactions. (Microsomal ALDH, which was used as a specificity control, does not really rule out this possibility since it is mainly cytosolic and exposes only four amino acids on the lumenal side of the ER (Masaki et al, 1994), whereas the major extramembranous parts of Gaa1p and Gpi8p are lumenal.) However, our data clearly indicate that the complex exists under physiological conditions, not only because of data obtained by blue native gel electrophoresis, but also because the complex could be purified from cells, in which its constitutent proteins were under the control of their physiological promoters: GPI16 and GAA1 were transcribed from their normal genomic gene; GPI8 was deleted but present on a centromeric vector as a GST-tagged recombinant protein.…”

Requirement of PIG-F and PIG-O for Transferring Phosphoethanolamine to the Third Mannose in Glycosylphosphatidylinositol

“…FLAG-tagged PIG-F was cotransfected with GST-tagged Pig-o, ALDH, and Pig-n separately into CHO cells. ALDH (38) and Pig-n (16) are ER membrane proteins. After the membranes had been dissolved with 1% digitonin, the GST-tagged proteins were precipitated with glutathione beads, and coprecipitation of FLAG-tagged proteins was analyzed by Western blotting (Fig.…”