High impact information on SEC12

• In yeast a type II integral membrane glycoprotein that is essential for transport vesicle budding from the endoplasmic reticulum (ER) is encoded by SEC12 (refs 1-3) [1].
• SEC12 encodes a guanine-nucleotide-exchange factor essential for transport vesicle budding from the ER [1].
• We show here that a purified fragment of Sec12 promotes guanine-nucleotide dissociation from Sar1 whereas the purified mutant Sec12-1 has only 15% of the wild-type activity [1].
• Large liposomes containing bound cytoplasmic domains of the v-SNAREs, Sec22p or Bos1p, or of the ER resident proteins, Sec12p and Ufe1p, were exposed to COPII proteins and GMP-PNP. v-SNAREs but not resident proteins were concentrated in synthetic COPII vesicles generated from donor liposomes [2].
• Furthermore, mitotic exit was sufficient to cause Chs2p neck localization specifically by triggering the Sec12p-dependent transport of Chs2p out of the ER [3].

Biological context of SEC12

• In the yeast secretory pathway, two genes SEC12 and SAR1, which encode a 70-kD integral membrane protein and a 21-kD GTP-binding protein, respectively, cooperate in protein transport from the ER to the Golgi apparatus [4].
• Second, Sec12p was shown to be essential for autophagy but not for the cytoplasm to vacuole-targeting (Cvt) (pathway, which shares mostly the same machinery with autophagy [5].
• This sorting is not blocked by a temperature-sensitive mutation in SEC12, which inhibits ER to Golgi transport, a null mutation in VPS8, which inhibits Golgi to PVC transport, or temperature-sensitive and null mutations in END4, which inhibit endocytosis from the plasma membrane [6].
• Glycosylation of Sec12p during biogenesis is indicated by an electrophoretic mobility shift of the protein that is influenced by tunicamycin and by imposition of an independent secretory pathway block [7].
• The formation of diffusible vesicles from the ER requires ATP, Sec12p, Sec23p, and GTP hydrolysis [8].

Anatomical context of SEC12

• First, the membranes from the sec12 mutant cells reproduce temperature sensitivity in the in vitro ER-to-Golgi transport reaction [4].
• Vesicle budding and protein transport from ER membranes containing normal levels of Sec12p is inhibited in vitro by addition of microsomes isolated from a Sec12p-overproducing strain [9].
• First, overexpression of the Sar1p-specific guanosine nucleotide exchange factor Sec12p was shown to result in the titration of the GTPase Sar1p, which is essential for COPII-coated vesicle formation [10].

Associations of SEC12 with chemical compounds

• Both proteins have NH2-terminal cytoplasmic domains which resemble that of Sec12p: they are similar in size and present a significant degree of amino acid identity with the cytoplasmic domain of Sec12p [11].
• Heat inactivation and trypsin treatment of cytosol, as well as addition of ATP gamma S to the chase incubations, led to a stabilization of p alpha F. ERAD was observed in sec12 microsomes, indicating that export of p alpha F via transport vesicles was not required [12].
• When the exit of vesicles from the endoplasmic reticulum or entry into the cis-Golgi were blocked in sec12 or sec18 cells, all anchors contained a side chain consisting of a single alpha 1,2-linked mannose [13].

Physical interactions of SEC12

• Sec12p is an ER type II membrane protein that mediates the membrane attachment of the GTP-binding Sar1 protein [11].
• We present the first evidence that Rer1p directly interacts with the transmembrane domain (TMD) of Sec12p which contains a retrieval signal [14].

Regulatory relationships of SEC12

• In vivo, the elevation of the SAR1 dosage suppresses temperature sensitivity of the sec12 mutant [4].
• To investigate whether Emp47p undergoes retrograde transport from the Golgi to the ER like other di-lysine-tagged proteins we developed an assay to measure this step after block of forward transport in a sec12 mutant [15].

Other interactions of SEC12

• The SAR1 gene is a multi-copy suppressor of a thermosensitive sec12 mutation [11].
• Sec12p, a close homologue of Sed4p, also acts early in the assembly of transport vesicles [16].
• In the delta(sed4) background, the suppression activity of SAR1 towards sec12 and sec16 is lost [17].
• In contrast, the lumenal domains of Sec12p, Stl1p and Stl2p are very different in size and do not show any appreciable homology [11].
• Pulse-chase experiments using these mutants show that alpha-COP and Rer1p function together in a very early Golgi compartment to initiate the recycling of Sec12p-derived hybrid proteins [18].

Analytical, diagnostic and therapeutic context of SEC12

• This assay employs membranes prepared from wild-type cells and cytosol fractions depleted of Sar1p due to overproduction of Sec12p or by gel filtration chromatography [19].
• Pulse-chase experiments and electron microscopy have revealed that the overproduction of Sec12p inhibits protein transport from the ER to the Golgi apparatus [20].
• Even in the rer1 disrupted cells, immunofluorescence of Sec12p stains the ER, implying that the retention system is still operating in the mutant [21].

References