Single Protein Tracking of P450-Reductase in an Endoplasmic Reticulum Biomimetic Reveals a NADPH Dependent Interaction with the Membrane

Some xenobiotics induce membrane-bound drug metabolizing enzymes (Xme) and a profound proliferation of the endoplasmic reticulum (ER) in vivo. However these effects are much weaker in vitro, possibly due to absence of certain transcription factors. We tested the possibility that ER proliferation can affect the level of ER-resident enzymes even in the absence of transcriptional activation. For this purpose we analysed the effects of compactin, which has been shown to induce ER proliferation in vitro, on recombinant Xme, which were expressed from a constitutive viral promoter. High levels of recombinant UDP-glucuronosyltransferase UGT1A6 were achieved by amplification of the UGT1A6 cDNA using the dihydrofolate reductase cDNA as selectable marker in DHFR- CHO cells. Treatment of the resulting cell lines with lipoprotein-deficient serum in the absence and presence of compactin for 5 days resulted in a 1.3- and 2.3-fold, respectively, increase of the UGT enzyme activity towards 4-methylumbelliferone, paralleled by an induction of immunoreactive UGT1A6 protein. Similarly, treatment with this 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor increased the endogenous P450 reductase activity 2.6-fold, concomitant with an increase of immunodetectable protein. As expected compactin induced the level of 3-hydroxy-3-methylglutaryl-CoA reductase. Increased levels of this protein have been associated with a proliferation of the ER. Compactin treatment of a separate cell line that expressed recombinant human P450 reductase increased this enzyme activity fivefold. Pulse-chase experiments revealed that the induction of the recombinant Xme by compactin was most likely due to decreased protein degradation. Our results show that enzyme systems unrelated to those involved in cholesterol biosynthesis are affected by compounds known to affect membrane biogenesis. Since this effect extends to heterologously expressed enzymes, it also provides an efficient means by which to increase the levels of recombinant ER proteins.

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PDZD8 mediates a Rab7-dependent interaction of the ER with late endosomes and lysosomes

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1913509116 ◽

2019 ◽

Vol 116 (45) ◽

pp. 22619-22623 ◽

Cited By ~ 19

Author(s):

Andrés Guillén-Samander ◽

Xin Bian ◽

Pietro De Camilli

Keyword(s):

Endoplasmic Reticulum ◽

Membrane Protein ◽

Hot Spots ◽

Transport Proteins ◽

Lipid Transport ◽

Domain Family ◽

Late Endosomes ◽

Dependent Interaction

Contacts between the endoplasmic reticulum (ER) and other membranes are hot spots for protein-mediated lipid transport between the 2 adjacent bilayers. Compiling a molecular inventory of lipid transport proteins present at these sites is a premise to the elucidation of their function. Here we show that PDZD8, an intrinsic membrane protein of the ER with a lipid transport module of the SMP domain family, concentrates at contacts between the ER and late endosomes/lysosomes, where it interacts with GTP-Rab7. These findings suggest that PDZD8 may cooperate with other proteins that function at the ER–endo/lysosome interface in coordinating endocytic flow with lipid transport between endocytic membranes and the ER.

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Demonstration that endoplasmic reticulum-associated degradation of glycoproteins can occur downstream of processing by endomannosidase

Biochemical Journal ◽

10.1042/bj20110186 ◽

2011 ◽

Vol 438 (1) ◽

pp. 133-142 ◽

Cited By ~ 13

Author(s):

Nikolay V. Kukushkin ◽

Dominic S. Alonzi ◽

Raymond A. Dwek ◽

Terry D. Butters

Keyword(s):

Quality Control ◽

Endoplasmic Reticulum ◽

Chinese Hamster ◽

Human Embryonic Kidney ◽

Previous Evidence ◽

Endoplasmic Reticulum Associated Degradation ◽

Single Protein ◽

Free Oligosaccharides ◽

Intermediate Compartment ◽

Functional Version

During quality control in the ER (endoplasmic reticulum), nascent glycoproteins are deglucosylated by ER glucosidases I and II. In the post-ER compartments, glycoprotein endo-α-mannosidase provides an alternative route for deglucosylation. Previous evidence suggests that endomannosidase non-selectively deglucosylates glycoproteins that escape quality control in the ER, facilitating secretion of aberrantly folded as well as normal glycoproteins. In the present study, we employed FOS (free oligosaccharides) released from degrading glycoproteins as biomarkers of ERAD (ER-associated degradation), allowing us to gain a global rather than single protein-centred view of ERAD. Glucosidase inhibition was used to discriminate between glucosidase- and endomannosidase-mediated ERAD pathways. Endomannosidase expression was manipulated in CHO (Chinese-hamster ovary)-K1 cells, naturally lacking a functional version of the enzyme, and HEK (human embryonic kidney)-293T cells. Endomannosidase was shown to decrease the levels of total FOS, suggesting decreased rates of ERAD. However, following pharmacological inhibition of ER glucosidases I and II, endomannosidase expression resulted in a partial switch between glucosylated FOS, released from ER-confined glycoproteins, to deglucosylated FOS, released from endomannosidase-processed glycoproteins transported from the Golgi/ERGIC (ER/Golgi intermediate compartment) to the ER. Using this approach, we have identified a previously unknown pathway of glycoprotein flow, undetectable by the commonly employed methods, in which secretory cargo is targeted back to the ER after being processed by endomannosidase.

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