The levels of endoplasmic reticulum proteins and ATP affect folding and secretion of selective proteins.
Proteins transiting the endoplasmic reticulum (ER) interact with a number of lumenal proteins, such as the glucose regulated proteins (GRPs), that either facilitate or prohibit protein folding and transport out of the ER compartment. We compared the relative amounts of mRNA encoding lumenal ER proteins in cells that secrete high levels of protein to those that do not secrete significant levels of protein. One of these proteins, GRP78, is thought to act as a chaperone to assist protein folding. We evaluated the effect of altered GRP78 expression on the secretion efficiency of heterologous proteins expressed in CHO cells. The secretion efficiency of proteins detected in significant association with GRP78 was reduced when GRP78 levels were overexpressed and improved when GRP78 levels were reduced. The results suggest that GRP78 does not act in a positive manner to promote protein folding and/or secretion. In addition, proteins that interact with GRP78 displayed a unique high requirement for intracellular ATP for secretion. Expression of firefly luciferase in the lumen of the ER detected ATP in the ER lumen of intact cells as monitored by light emission. Since luciferase light emission is proportional to ATP concentration, the amount of light emission may provide an approach to study the effect of altered ER intralumenal ATP on protein folding and secretion.
Dorner
A J
AJ
Genetics Institute, Cambridge, Massachusetts.
Kaufman
R J
RJ
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001688:Biological Products / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D001693:Biological Transport, Active; D016466:CHO Cells; D002352:Carrier Proteins / Q000378:metabolism; D002451:Cell Compartmentation; D002460:Cell Line; D006224:Cricetinae; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D000091342:Endoplasmic Reticulum Chaperone BiP; D005169:Factor VIII / Q000096:biosynthesis; D006023:Glycoproteins / Q000378:metabolism; D006360:Heat-Shock Proteins; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D018832:Molecular Chaperones; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D001688:Biological Products; D002352:Carrier Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; D006023:Glycoproteins; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D011506:Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D005169:Factor VIII; D008156:Luciferases