Differential fates of invertase mutants in the yeast endoplasmic reticulum

Mutation of the protein-O-mannosyltransferase enhances secretion of the human urokinase-type plasminogen activator inHansenula polymorpha

“…It is known that in many cases the rate‐limiting step in secretion of heterologous proteins in S. cerevisiae occurs at the ER‐to‐Golgi transition, and the efficiency of their secretion is determined by the capacity of the protein‐folding machinery in the ER (Parekh and Wittrup, 1997; Kowalski et al , 1998; McCracken et al , 2000). Human uPA expressed in S. cerevisiae and H. polymorpha has also been shown to be retained in the ER (Hiramatsu et al , 1991; Agaphonov et al , 2002).…”

“…Although organization of the secretory pathway in yeast has the same basic principles as in other eukaryotic organisms, not all proteins of higher eukaryotes can be efficiently secreted from their cells. Secretion of some recombinant or mutant proteins is hampered by inefficient folding in the yeast endoplasmic reticulum (ER) (Parekh and Wittrup, 1997; Holkeri and Makarow, 1998; McCracken et al , 2000), which, in turn, causes their rapid degradation by the cytosolic proteasome complex after retrograde translocation from the lumenal to the cytoplasmic face of the ER, a mechanism called ‘ER‐associated degradation’ (ERAD; Hiller et al , 1996). The recognition and retention of misfolded proteins in the ER is carried out by the so‐called ‘ER quality control’ machinery (for review, see Parodi, 1999).…”

Fusion of an intact secretory protein permits a misfolded protein to exit from the endoplasmic reticulum in yeast

“…Although the E486 K mutation is unlikely to destabilize invertase, 40) we cannot exclude the possibility that it was retained in the ER by quality control mechanisms due to a conformational change in the invertase domain rather than a loss of interactions with the corresponding receptors.…”

“…38) Although the ER exit signal within invertase has not yet been identified, it has been reported that glutamate-486 was involved in its exit from the ER. 39,40) This amino acid residue is highly conserved among members of glycoside hydrolase Note: Cell lysates were prepared from prc1Δhrd1Δ pep4Δ (YKS30) cells expressing CPY (A), CPY* (A), CPY-Inv (B), or CPY*-Inv (B), and then incubated with or without trypsin for the indicated times, as described in "Materials and methods." After Endo H treatment, samples were analyzed by SDS-PAGE, followed by immunoblot analysis with anti-CPY antiserum.…”

“…41) Replacement of the glutamate-486 residue with lysine in yeast invertase caused retention of it in the ER without noticeable degradation. 39,40) Hence, we decided to introduce this mutation into CPY*-Inv to test the possibility that the invertase domain can function as the ER exit signal for CPY*-Inv. When CPY*-Inv E486 K was expressed in wild-type cells, it exhibited slight stabilization as compared with CPY*-Inv (Fig.…”