N-terminal domain of Gpa1 (G protein alpha) subunit) is sufficient for plasma membrane targeting in yeast Saccharomyces cerevisiae

1998 ◽  
Vol 111 (21) ◽  
pp. 3235-3244
Author(s):  
K.M. Gillen ◽  
M. Pausch ◽  
H.G. Dohlman
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
G Protein ◽  
Alpha Subunit ◽  
Membrane Targeting ◽  
Yeast Saccharomyces Cerevisiae ◽  
N Terminus ◽  
Membrane Attachment ◽  
G Protein Alpha Subunit ◽  
Protein Alpha Subunit

G proteins play a central role in transmitting signals from cell surface receptors to effector proteins inside the cell. Signaling can only occur, however, if all these protein components are properly assembled and localized at the plasma membrane. Past studies have shown that certain segments within the N-terminal region of the G protein alpha subunit are necessary for membrane attachment. Here we identify a region within the yeast G alpha (Gpa1) that is sufficient for membrane attachment, as well as for specific targeting to the plasma membrane. Initially, we constructed chimeric proteins that replace the N terminus of mammalian Gsalpha with the corresponding sequence from Gpa1. Gsalpha is inefficiently targeted to the yeast plasma membrane and therefore cannot fully complement the loss of Gpa1. Gpa1-Gsaplha chimeras were assayed for proper membrane localization by functional complementation of a gpa1Delta;) mutant, and by sucrose density gradient fractionation of cell membranes. Most of the chimeras tested, including one with only the N-terminal 7 amino acids from Gpa1, exhibited normal membrane targeting and complementing activity. We also fused various lengths of N-terminal Gpa1 sequence to glutathione-S-transferase (GST), a heterologous protein normally expressed in the cytoplasm. The first 67- 36- or 9-amino acids of Gpa1 were all sufficient to direct GST specifically to the plasma membrane in yeast. This analysis defines the extreme N terminus of Gpa1 as the primary determinant of proper membrane targeting, and represents an essential step towards isolating and identifying G protein-targeting proteins within the plasma membrane.

scholarly journals Isolation and expression of a novel chick G-protein cDNA coding for a Gαi3 protein with a Gα0 N-terminus

1994 ◽  
Vol 297 (2) ◽  
pp. 303-308 ◽  
Author(s):  
E J Kilbourne ◽  
J B Galper
Keyword(s):  
G Protein ◽  
Sequence Similarity ◽  
In Vitro Transcription ◽  
Alpha Subunit ◽  
Alpha Subunits ◽  
N Terminus ◽  
Brain Cdna Library ◽  
G Protein Alpha Subunit ◽  
Protein Alpha Subunit

We have cloned cDNAs coding for G-protein alpha subunits from a chick brain cDNA library. Based on sequence similarity to G-protein alpha subunits from other eukaryotes, one clone was designated G alpha i3. A second clone, G alpha i3-o, was identical to the G alpha i3 clone over 932 bases on the 3′ end. The 5′ end of G alpha i3-o, however, contained an alternative sequence in which the first 45 amino acids coded for are 100% identical to the conserved N-terminus of G alpha o from species such as rat, mouse, human, bovine and hamster. Both clones were found to be expressed in all tissues studied. The unusual alpha o-alpha i3-like G-protein chimera, G alpha i3-o, was found to be expressed at significantly lower levels than G alpha i3. In vitro transcription and translation of the G alpha i3-o cDNA clone gave a protein of approx. 41 kDa which stably bound guanosine 5′-[gamma-thio]triphosphate. G alpha i3-o appears to be the first G-protein alpha subunit cloned which contains ends that are homologous to two different alpha subunit isoforms, G alpha o and G alpha i3.

scholarly journals Cloning and targeted mutations of G alpha 7 and G alpha 8, two developmentally regulated G protein alpha-subunit genes in Dictyostelium.

1994 ◽  
Vol 5 (6) ◽  
pp. 691-702 ◽  
Author(s):  
L Wu ◽  
C Gaskins ◽  
K Zhou ◽  
R A Firtel ◽  
P N Devreotes
Keyword(s):  
Amino Acids ◽  
G Protein ◽  
Alpha Subunit ◽  
Hydrolysis Rate ◽  
Complete Analysis ◽  
Developmental Defects ◽  
Alpha Subunits ◽  
Alpha 7 ◽  
G Protein Alpha Subunit ◽  
Protein Alpha Subunit

GTP-binding protein (G protein)-mediated signal transduction pathways play essential roles during the aggregation and differentiation process of Dictyostelium. In addition to the five known G protein alpha-subunit genes, we recently identified three novel alpha-subunit genes, G alpha 6, G alpha 7, and G alpha 8, using the polymerase chain reaction technique. We present here a more complete analysis of G alpha 7 and G alpha 8. The cDNAs of these two genes were cloned, and their complete nucleotide sequences were determined. Sequence analyses indicate that G alpha 8 possesses some unusual features. It lacks the "TCATDT" motif, a sequence of amino acids highly conserved among G alpha subunits, and has an additional 50 amino acids at its C-terminus consisting of long stretches of asparagine. Moreover, G alpha 8 is unusually resistant to protease digestion, which may indicate a slow GTP hydrolysis rate. The possible functions of these alpha-subunits were assessed by generating mutants lacking G alpha 7 or G alpha 8 by gene targeting through homologous recombination and by overexpressing G alpha 7 or G alpha 8 protein. Overexpression of G alpha 7 resulted in abnormal morphogenesis starting at the slug stage, whereas analysis of the other strains failed to reveal any obvious growth or developmental defects under either normal or stressful conditions. The implications of these results are discussed.

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