Kex2-dependent invertase secretion as a tool to study the targeting of transmembrane proteins which are involved in ER-->Golgi transport in yeast.
Mutants were isolated that are defective in the retention of a transmembrane protein in the early secretory compartments in yeast. A series of hybrid proteins was tested for their use in the selection of such mutants. Each of these hybrid proteins consisted of a type II transmembrane protein (Nin/Cout) and invertase (Suc2) as a reporter separated by a peptide linker containing a cleavage site for the Golgi protease Kex2. The integral membrane proteins which were used--Sec12p, Sec22/Sly2p or Bet1/Sly12p--are all known to be required for ER-->Golgi transport in yeast. Invertase was readily cleaved from the fusions containing Sec22/Sly2p or Bet1/Sly12p as the membrane anchoring part. In contrast, Sec12--invertase expressing transformants required mutations in either of two different genes for Kex2-dependent invertase secretion. The mutant showing the stronger retention defect (rer1) was used to clone the corresponding gene. RER1 represents the first reading frame left of the centromere of chromosome III. Cells carrying a disruption of the RER1 gene are viable and show the same mislocalizing phenotype as the original mutants. The Rer1 protein, as deduced from the nucleotide sequence, contains four transmembrane domains. It has been suggested before that Sec12p cycles between the ER and the cis-Golgi compartment. Some results obtained by using Sec12-invertase and the rer1 mutants resemble observations on the retention of Golgi-resident glycosyltransferases and viral proteins in mammalian cells. For instance, retention of Sec12-invertase is non-saturable and the membrane-spanning domain of Sec12p seems to constitute an important targeting signal.
Boehm
J
J
Department of Molecular Genetics, Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany.
Ulrich
H D
HD
Ossig
R
R
Schmitt
H D
HD
D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D005800:Genes, Fungal; D017930:Genes, Reporter; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism*; D016695:Glycosyltransferases / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D015388:Organelles / Q000378:metabolism*; D043484:Proprotein Convertases; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013381:Subtilisins / Q000378:metabolism*; D033921:Vesicular Transport Proteins; D014764:Viral Proteins / Q000378:metabolism; D043324:beta-Fructofuranosidase
D005656:Fungal Proteins; D020662:Guanine Nucleotide Exchange Factors; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; C089137:RER1 protein, S cerevisiae; D011993:Recombinant Fusion Proteins; C071239:SEC12 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; D014764:Viral Proteins; D016695:Glycosyltransferases; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase; D043484:Proprotein Convertases; D013381:Subtilisins; C058751:KEX2 protein, S cerevisiae