Localization and targeting of the Saccharomyces cerevisiae Kre2p/Mnt1p alpha 1,2-mannosyltransferase to a medial-Golgi compartment.
The yeast Kre2p/Mnt1p alpha 1,2-mannosyltransferase is a type II membrane protein with a short cytoplasmic amino terminus, a membrane-spanning region, and a large catalytic luminal domain containing one N-glycosylation site. Anti-Kre2p/Mnt1p antibodies identify a 60-kD integral membrane protein that is progressively N-glycosylated in an MNN1-dependent manner. Kre2p/Mnt1p is localized in a Golgi compartment that overlaps with that containing the medial-Golgi mannosyltransferase Mnn1p, and distinct from that including the late Golgi protein Kex1p. To determine which regions of Kre2p/Mnt1p are required for Golgi localization, Kre2p/Mnt1p mutant proteins were assembled by substitution of Kre2p domains with equivalent sequences from the vacuolar proteins DPAP B and Pho8p. Chimeric proteins were tested for correct topology, in vitro and in vivo activity, and were localized intracellularly by indirect immunofluorescence. The results demonstrate that the NH2-terminal cytoplasmic domain is necessary for correct Kre2p Golgi localization whereas, the membrane-spanning and stem domains are dispensable. However, in a test of targeting sufficiency, the presence of the entire Kre2p cytoplasmic tail, plus the transmembrane domain and a 36-amino acid residue luminal stem region was required to localize a Pho8p reporter protein to the yeast Golgi.
D000595:Amino Acid Sequence; D001483:Base Sequence; D002451:Cell Compartmentation / Q000502:physiology; D004721:Endoplasmic Reticulum / Q000201:enzymology; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D006056:Golgi Apparatus / Q000201:enzymology*; D008364:Mannosyltransferases / Q000737:chemistry / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000737:chemistry; D014176:Protein Biosynthesis / Q000502:physiology; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D005656:Fungal Proteins; D008565:Membrane Proteins; D015345:Oligonucleotide Probes; D011993:Recombinant Fusion Proteins; D008364:Mannosyltransferases