The GET Complex Mediates Insertion of Tail-Anchored Proteins into the ER Membrane

Summary Tail-anchored (TA) proteins, defined by the presence of a single C-terminal transmembrane domain (TMD), play critical roles throughout the secretory pathway and in mitochondria, yet the machinery responsible for their proper membrane insertion remains poorly characterized. Here we show that Get3, the yeast homolog of the TA-interacting factor Asna1/Trc40, specifically recognizes TMDs of TA proteins destined for the secretory pathway. Get3 recognition represents a key decision step, whose loss can lead to misinsertion of TA proteins into mitochondria. Get3-TA protein complexes are recruited for endoplasmic reticulum (ER) membrane insertion by the Get1/Get2 receptor. In vivo, the absence of Get1/Get2 leads to cytosolic aggregation of Get3-TA complexes and broad defects in TA protein biogenesis. In vitro reconstitution demonstrates that the Get proteins directly mediate insertion of newly synthesized TA proteins into ER membranes. Thus, the GET complex represents a critical mechanism for ensuring efficient and accurate targeting of TA proteins.

resulting strain was used for co-purification of both proteins. After preculturing in selective medium the yeast strain was grown in 2l YPD media to final OD 600 =1.5-1.8 which resulted in approximately 20g of cells. Cells were collected by centrifugation for 10min at 4500g, washed once with water, shock frozen in liquid nitrogen and stored at -80°C.
Library plasmid DNA from colonies displaying activity of all three reporter genes (HIS3, ADE2, lacZ) was rescued (Ausubel et al., 1997) and retransformed with the original bait construct and a GFP-containing control construct. Transformants were tested for the baitspecific activity of the HIS3 and ADE2 reporter genes and the corresponding plasmids were sequenced. For testing specific yeast two-hybrid constructs strain AH109 (Clontech) was transformed with the constructs indicated in the figure and plated on dropout medium lacking tryptophane and leucine to recover transformants. Individual colonies were grown in liquid culture and serial dilutions were spotted on dropout medium lacking tryptophane and leucine or lacking tryptophane, leucine, and histidine.

Fluorescence microscopy
For Figure  For the supplementary figures cells were analyzed by multiple wavelength fluorescence and visible light microscopy with a digital imaging capable Nikon TE200/300 inverted microscopy using an oil-immersed objective at X100 magnification.

Kar2 Secretion Assays
Cells were grown in YEPD at 30°C to mid-log phase, harvested, resuspended in fresh medium to OD 600 =0.5 and incubated for 2h. Proteins were precipitated from 1.35ml of supernatants by addition of TCA to 10%. The pellet was washed with acetone, resuspended in 35µl of 1x SDS-PAGE sample buffer and neutralized with 1mM Tris pH=9.4. Proteins were subjected to SDS-PAGE, transferred to a nitrocellulose membrane and analyzed by immunoblotting using antisera against Kar2 (1:5000), (Kind gift from Peter Walter). Anti-rabbit secondary antibody conjugated to IRdye800 (Rockland) (used at 1:10,000) was detected using Odyssey (Li-COR) fluorescent scanner and software.

Preparation of Microsomes
Membranes were prepared from cells grown to OD 600 1-2 in YPD by spheroplast lysis.

Co-Immunoprecipitation
Yeast cells were grown to an OD 600 =0.5 in the appropriate dropout medium lacking leucine and uracil and containing 2% glucose as the carbon source. 150 OD 600 units were harvested and broken in 500µl breaking buffer (20mM HEPES pH 7.4, 100mM KCl, 2×Complete protease inhibitor cocktail; Roche) using glass beads. The resulting extract was diluted 1:1 with immunoprecipitation buffer (50mM Tris-HCl pH 7.6, 150mM NaCl, 1mM EDTA, and 1% NP-40, complemented with Complete protease inhibitor) and incubated with 5µg mouse monoclonal anti-HA (HA.11, Covance) and protein G-sepharose (Amersham) overnight, washed three times in immunoprecipitation buffer and several times in PBS, and eluted in SDS-PAGE loading buffer without reducing agent at 37°C.

Whole Cell Extracts and Immunoblotting
Cells were grown in SD dropout medium at 30°C to mid-log phase, then transferred to a 1.5ml reaction tube and centrifuged for 5min at 4,000 rpm. The pellet was resuspended in Washes were in TBS-blocking solution and then in TBS, 0.02% NP-40. Detection was performed using the ECL system (Amersham) or an Odyssey (Li-COR) fluorescent scanner.

Subcellular Fractionation on Sucrose Gradients
Separation of organelles was performed as described {Nass, 1998 #73}. Briefly, cells were grown to mid-logarithmic phase, converted to spheroblasts and lysed by douncing in a hypotonic buffer (0.3M sorbitol, 50mM triethanol amine pH 8.9). The homogenate was layered on freshly prepared ten-step sucrose gradients and centrifuged for 12h at 23,500rpm in an SW28 rotor (Beckman). Membranes from the different fractions were pelleted by centrifugation (SW28, 30minutes at 23,500rpm). 10µg of membrane protein was loaded per fraction and resolved by SDS-PAGE.

Triton-Solubility Assay Employing Subcellular Fractions
10µg protein of pellets obtained from the dense sucrose fractions 8 and 9 (46% and 50%) were incubated for 10minutes in 20µl 20mM HEPES, 1% Triton-X100 at 30°C. After a 15min 16,000rcf centrifugation step, 4µl 5xSDS-PAGE sample buffer and 100mM DTT were added to the supernatant. The pellet was resuspended in 25µl 1xSDS-PAGE sample buffer including 100mM DTT. 15µl of each pellet and supernatant were subjected to SDS-PAGE, transferred to a nitrocellulose membrane and analyzed by immunoblotting using antiserum against Get3.

Protease protection for in vitro translocation assays
Following translocation, the reactions were treated with 0.25mg/ml Proteinase K (Worthington Biochemical Company) in either the presence or the absence of 1% Triton X-100 on ice for 30min. PMSF was then added (10mM) followed by further incubation on ice for 5min. Finally, boiling loading buffer was added and the samples were kept boiling for 5min to ensure that Proteinase K is irreversibly inactivated.