A microsomal GTPase is required for glycopeptide export from the mammalianendoplasmic reticulum

Bidirectional transport of proteins via the Sec61p translocon across the en doplasmic reticulum (ER) membrane is a recognized component of the ER quali ty control machinery. Following translocation and engagement by the luminal quality control system, misfolded and unassembled proteins are exported fr om the ER lumen back to the cytosol for degradation by the proteasome. Addi tionally, other ER contents, including oligosaccharides, oligopeptides, and glycopeptides, are efficiently exported from mammalian and yeast systems, indicating that bidirectional transport across ER membranes is a general eu karyotic phenomenon Glycopeptide and protein export from the ER in in vitro systems is both ATP- and cytosol-dependent. Using a well established syste m to study glycopeptide export and conventional liquid chromatography, we i solated a single polypeptide species of 23 kDa from rat Liver cytosol that was capable of fully supporting glycopeptide export from rat microsomes in the presence of an ATP-regenerating system. The protein was identified by m ass spectrometric sequence analysis as guanylate kinase (GK), a housekeepin g enzyme critical in the regulation of cellular GTP levels. We confirmed th e ability of GK to substitute for complete cytosol by reconstitution of gly copeptide export from rat Liver microsomes using highly purified recombinan t GK from Saccharomyces cerevisiae. Most significantly, we found that the G K (and hence the cytosolic component) requirement was fully bypassed by low micromolar concentrations of GDP or GTP. Similarly, export was inhibited b y non-hydrolyzable analogues of GDP and GTP, indicating a requirement for G TP hydrolysis. Membrane integrity was fully maintained under assay conditio ns, as no ER luminal. proteins mere released. Competence for glycopeptide e xport was abolished by very mild protease treatment of microsomes, indicati ng the presence of an essential protein on the cytosolic face of the ER mem brane. These data demonstrate that export of glycopeptide export is control led by a microsomal GTPase and is independent of cytosolic protein factors.