HUMAN RER1 IS LOCALIZED TO THE GOLGI-APPARATUS AND COMPLEMENTS THE DELETION OF THE HOMOLOGOUS RER1 PROTEIN OF SACCHAROMYCES-CEREVISIAE

Secl2p is a type II membrane glycoprotein in the endoplasmic reticulum (ER) of Saccharomyces cerevisiae which is essential for transport ves icle budding, It is the guanine nucleotide exchange factor for the sma ll GTP-binding protein Sar1p which is a constituent of COP II ER to Go lgi vesicles, We report the sequence and localization of the human hom ologue to yeast Rer1p, which has recently been identified genetically as an essential component for retention of Secl2p in the ER, Reverse p olymerase chain reaction was used to obtain cDNAs from HeLa cells, The y code for a protein of 196 amino acids, corresponding to a molecular mass of 23 kDa, The translated sequence is 44 % identical and 65% simi lar to yeast Rer1 protein, The four putative transmembrane domains are predicted to form a W-topology with both N-and C-terminus facing the cytosol, The functional activity of myc-tagged human Rer1 was demonstr ated by the complementation of the RER1 deletion in S. cerevisiae. Mis localization of the Secl2 reporter protein was reduced similar to the results obtained with yeast Rer1p, Human Rer1 protein was expressed in HeLa cells and the subcellular distribution analyzed by double immuno fluorescence and immunoelectron microscopy of thawed cryosections. The tagged protein was localized to the Golgi apparatus and peripheral el ements of the ER-Golgi interface, High overexpression leads to relocat ion of human Rer1 to ER-like structures together with KDEL-receptor an d affects the structural organization of the Golgi apparatus, Under co nditions of brefeldin A treatment, human Rer1 distributes together wit h recycling Golgi proteins,Sec12p is a type II membrane glycoprotein i n the endoplasmic reticulum (ER) of Saccharomyces cerevisiae which is essential for transport vesicle budding, It is the guanine nucleotide exchange factor for the small GTP-binding protein Sar1p which is a con stituent of COP II ER to Golgi vesicles, We report the sequence and lo calization of the human homologue to yeast Rer1p, which has recently b een identified genetically as an essential component for retention of Sec12p in the ER, Reverse polymerase chain reaction was used to obtain cDNAs from HeLa cells, They code for a protein of 196 amino acids, co rresponding to a molecular mass of 23 kDa, The translated sequence is 44% identical and 65% similar to yeast Rer1 protein, The four putative transmembrane domains are predicted to form a W-topology with both N- and C-terminus facing the cytosol, The functional activity of myc-tag ged human Rer1 was demonstrated by the complementation of the RER1 del etion in S, cerevisiae. Mislocalization of the Sec12- reporter protein was reduced similar to the results obtained with yeast Rer1p, Human R er1 protein was expressed in HeLa cells and the subcellular distributi on analyzed by double immunofluorescence and immunoelectron microscopy of thawed cryosections. The tagged protein was localized to the Golgi apparatus and peripheral elements of the ER-Golgi interface, High ove rexpression leads to relocation of human Rer1 to ER-like structures to gether with KDEL-receptor and affects the structural organization of t he Golgi apparatus, Under conditions of brefeldin A treatment, human R er1 distributes together with recycling Golgi proteins.