Cloning, characterisation, and functional expression of the Mus musculus SKD1 gene in yeast demonstrates that the mouse SKD1 and the yeast VPS4 genes are orthologues and involved in intracellular protein trafficking.
The mouse SKD1 protein displays a high degree of sequence identity (62%) to the yeast Vps4 protein, which is involved in the transport of proteins out of a prevacuolar/endosomal compartment. We isolated the mouse SKD1 locus and found that the SKD1 gene is split into 11 exons covering a region of 29kb of the genome. Interestingly, the exon/intron structure reflects to a certain degree the proposed domain structure of the protein, since the 5' located coiled-coil region and the AAA domain are flanked by introns. Analysis of the promoter region, which revealed features common for 'housekeeping genes', is consistent with previous results of a mouse multi-tissue Northern blot, confirming that SKD1 is a ubiquitously expressed gene. Expression of the full-length SKD1 cDNA in a vps4 disrupted yeast strain suppressed the temperature-sensitive growth defect of the vps4 mutant strain. Overexpression of wild type and expression of mutant Vps4 and SKD1 proteins, harbouring single amino acid exchanges in their AAA domains, induced a dominant-negative vacuolar protein sorting defect in wild type yeast cells, indicating that mouse SKD1 protein and yeast Vps4p fulfil similar functions.
Biologisch-Medizinisches Forschungszentrum, Heinrich-Heine-Universitat Dusseldorf, Moorenstrasse 5, 40225, Dusseldorf, Germany.
S
S
Scheuring
O
O
Bodor
R A
RA
Röhricht
S
S
Müller
A
A
Beyer
K
K
Köhrer
D000074183:ATPases Associated with Diverse Cellular Activities; D000251:Adenosine Triphosphatases / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D056827:Endosomal Sorting Complexes Required for Transport; D005656:Fungal Proteins / Q000235:genetics*; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011506:Proteins / Q000378:metabolism*; D012097:Repressor Proteins / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D012689:Sequence Homology, Nucleic Acid; D013696:Temperature; D033921:Vesicular Transport Proteins
D004274:DNA, Recombinant; D056827:Endosomal Sorting Complexes Required for Transport; D005656:Fungal Proteins; D011506:Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; C107528:VPS4 protein, S cerevisiae; D033921:Vesicular Transport Proteins; D000251:Adenosine Triphosphatases; D000074183:ATPases Associated with Diverse Cellular Activities; C490304:VPS4B protein, human; C089217:Vps4b protein, mouse