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. 1999 Dec;10(12):4263-81.
doi: 10.1091/mbc.10.12.4263.

The yeast GRD20 gene is required for protein sorting in the trans-Golgi network/endosomal system and for polarization of the actin cytoskeleton

R G Spelbrink  1 , S F Nothwehr
Affiliations
Free PMC article

The yeast GRD20 gene is required for protein sorting in the trans-Golgi network/endosomal system and for polarization of the actin cytoskeleton

R G Spelbrink et al. Mol Biol Cell. 1999 Dec.
Free PMC article

Abstract

The proper localization of resident membrane proteins to the trans-Golgi network (TGN) involves mechanisms for both TGN retention and retrieval from post-TGN compartments. In this study we report identification of a new gene, GRD20, involved in protein sorting in the TGN/endosomal system of Saccharomyces cerevisiae. A strain carrying a transposon insertion allele of GRD20 exhibited rapid vacuolar degradation of the resident TGN endoprotease Kex2p and aberrantly secreted approximately 50% of the soluble vacuolar hydrolase carboxypeptidase Y. The Kex2p mislocalization and carboxypeptidase Y missorting phenotypes were exhibited rapidly after loss of Grd20p function in grd20 temperature-sensitive mutant strains, indicating that Grd20p plays a direct role in these processes. Surprisingly, little if any vacuolar degradation was observed for the TGN membrane proteins A-ALP and Vps10p, underscoring a difference in trafficking patterns for these proteins compared with that of Kex2p. A grd20 null mutant strain exhibited extremely slow growth and a defect in polarization of the actin cytoskeleton, and these two phenotypes were invariably linked in a collection of randomly mutagenized grd20 alleles. GRD20 encodes a hydrophilic protein that partially associates with the TGN. The discovery of GRD20 suggests a link between the cytoskeleton and function of the yeast TGN.

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Figures

Figure 1
Figure 1
The GRD20 encodes a conserved protein with a predicted coiled-coil domain. (A) Whole-cell protein extracts from strains SNY17 (GRD20), SBY7-7A (grd20::Tn3), and SBY4-10A (grd20-Δ1) were separated by SDS-PAGE and analyzed by Western blot using a rabbit anti-Grd20p antibody. (B) Drawings representing the Grd20p and Y71F9A-290.a protein sequences. The shaded boxes denote regions of sequence similarity between the two proteins (23.5% identity using the ALIGN program with default settings). The black box in Grd20p corresponds to a region (residues 87–114) having a high propensity for forming coiled-coil structure (p = 0.931 using COILS 2.1 program [Lupas et al., 1991] with a window of 28). The arrow indicates the approximate position of the Tn3 transposon insertion in the grd20::Tn3 allele.
Figure 2
Figure 2
A loss of Grd20p function causes a severe growth defect. The following strains were spotted onto YEPD plates from left to right: SNY17, SBY4-10A, SBY7-7A, SBY4-10A carrying pSB1, SBY4-10A carrying pSB1-125, and SBY4-10A carrying pSB1-1212 (see Table 2 for descriptions of plasmids). Approximately 5000, 500, and 50 viable cells were applied at the three positions (from top to bottom) for each strain. The plates were incubated at the indicated temperatures for 36 h before being photographed.
Figure 3
Figure 3
Grd20p is required for efficient sorting of the vacuolar hydrolase CPY. (A) Strains SNY17 (GRD20), SBY4-10A (grd20-Δ1), SNY97-14D (grd20::Tn3), and SBY4-10A/pSB10 (grd20-Δ1 + pSB10) were pulsed with [35S]methionine and cysteine for 10 min and chased with unlabeled amino acids for 45 min. The strains were incubated at 30°C throughout the experiment. CPY was immunoprecipitated from the intracellular (I) and extracellular (E) fractions and analyzed by SDS-PAGE and fluorography. The left panel was overexposed to allow detection of CPY in the grd20-Δ1 strain that incorporated label poorly. (B) The following strain/plasmid combinations were propagated for several doublings at 22°C: SBY4-10A/pSB1 (GRD20), SBY4-10A/pSB1-125 (grd20-1), and SBY4-10A/pSB1-1212 (grd20-2). The strains were then shifted to 36°C for 5 min or incubated at 22°C as indicated. After a 10-min pulse and 0- and 45-min chase, CPY was immunoprecipitated and analyzed as in A. (C) Strain SBY4-10A transformed with plasmids pSB1 + pSB18 (GRD20 + GRD20), pSB1 + pSN335 (GRD20 + grd20-1), pSB1 + pSN336 (GRD20 + grd20-2), and pSB1-125 + pSN336 (grd20-1 + grd20-2) was subjected to CPY immunoprecipitation as described in B after a 45-min chase. For both A and B the percentage of CPY secreted from each strain after a 45-min chase as determined by phosphorimager analysis is indicated below each E lane. The CPY secretion values in B represent the average of two experiments, and the values from each of those experiments are shown in Table 3 for strains incubated at 36°C. The positions of ER (p1) and Golgi (p2) precursor forms and vacuole localized mature form (m) of CPY indicated were assigned based on the migration of CPY expressed in wild-type strains.
Figure 4
Figure 4
Kex2p is rapidly degraded by vacuolar proteases in grd20 mutants. (A) Colony immunoblot of strains AHY47/pSN179-A (GRD20) and SBY9-8A/pSN179-A (grd20::Tn3) in which secreted, unprocessed α-factor was detected using a rabbit anti-α-factor antibody. (B) Strains SNY17 (GRD20), SBY7-7A (grd20::Tn3), and SBY6 (pep4Δ grd20::Tn3) were pulsed for 10 min and chased for the indicated times at 30°C. Kex2p was then immunoprecipitated and analyzed by SDS-PAGE and fluorography. (C) The following strain/plasmid combinations were propagated for several doublings at 22°C, shifted to 36°C for 5 min, and then pulsed for 10 min and chased for the indicated times: SBY4-10A/pSB1 (GRD20), SBY4-10A/pSB1-125 (grd20-1), SBY4-10A/pSB1-1212 (grd20-2), and SBY7-7A. After immunoprecipitation and separation by SDS-PAGE, the percentage of Kex2p remaining at each time point was determined and normalized to the amount of Kex2p present at 0 min (arbitrarily set at 100%).
Figure 5
Figure 5
Kex2p is mislocalized to the vacuole in grd20 mutants. GRD20 pep4Δ (LSY2) and grd20::Tn3 pep4Δ (SBY6) strains propagated at 30°C were fixed, spheroplasted, and costained with antibodies against Kex2p and Vma2p. After subsequent treatment with fluorochrome-conjugated antibodies, the cells were viewed by differential interference contrast optics and by epifluorescence through filters specific for FITC and Texas Red.
Figure 6
Figure 6
Localization and transport of A-ALP and ALP in grd20 mutants. Strains in A and B were propagated at 30°C, pulsed for 10 min, and chased for the indicated times, and either A-ALP or ALP was immunoprecipitated and analyzed by SDS-PAGE and fluorography. (A) Analysis of strains SNY17 (GRD20), SBY7-7A (grd20::Tn3), and SBY4-10A (grd20-Δ1) carrying a CEN plasmid directing expression of A-ALP (pSN55). (B) Analysis of strains SNY17 (GRD20) and SBY7-7A (grd20::Tn3) carrying a CEN plasmid directing expression of ALP (pSN92). The positions at which precursor A-ALP (pA-ALP), mature A-ALP (mA-ALP), precursor ALP (pALP), mature ALP (mALP), and a breakdown product of mature ALP (*) migrate are indicated. (C) Strains LSY2 (GRD20 pep4Δ) and SBY6 (grd20::Tn3 pep4Δ) carrying pSN55 were fixed, spheroplasted, and costained with antibodies against A-ALP (upper panels) and Vma2p (lower panels). After subsequent treatment with fluorochrome-conjugated antibodies, the cells were viewed by differential interference contrast optics and by epifluorescence through filters specific for FITC and Texas Red.
Figure 7
Figure 7
The CPY receptor Vps10p is only slightly unstable in grd20 mutants. Strains SNY17 (GRD20) and SNY97–14D (grd20::Tn3) were pulsed for 10 min and chased for the indicated times at 30°C. Vps10p was then immunoprecipitated and analyzed by SDS-PAGE and fluorography. The positions of intact Vps10p and a form (*) generated by proteolytic cleavage by vacuolar proteases (Cereghino et al., 1995) are indicated.
Figure 8
Figure 8
Invertase secretion kinetics and carbohydrate processing in grd20 mutants. Strains SNY17 (GRD20), SBY4-10A/pSB1-125 (grd20-1), and SBY7-7A (grd20::Tn3) carrying plasmid pSB19 were propagated at 22°C and then shifted to 36°C for 15 min before a 10-min pulse and 0- or 30-min chase. Invertase immunoprecipitated from intracellular (I) and extracellular (E) samples was then analyzed by SDS-PAGE and fluorography. The positions of the ER glycosylated and early Golgi modified forms of invertase (core) and the medial and late Golgi forms (outer chain) are indicated. The percent invertase secretion at each time point as determined by phosphorimager analysis is indicated below the E lanes for strains GRD20 and grd20-1. The values for percent secretion represent the average of two independent experiments.
Figure 9
Figure 9
Grd20p is necessary for normal actin polarization and organization. Strains SBY4-10A/pSB1 (GRD20) and SBY4-10A/pSB1-125 (grd20-1) were grown for several doublings at 22°C and then shifted to 36°C for 2 h, whereas strain SBY4-10A (grd20-Δ1) was propagated at 30°C before fixation. The cells were then fixed, stained with phalloidin conjugated to Texas Red, washed, mounted on slides, and visualized using differential interference contrast optics and by epifluorescence through a filter specific for Texas Red.
Figure 10
Figure 10
Grd20p is required for rapid endocytic delivery of Ste3p to the vacuole. The following strain/plasmid combinations were grown for several generations at 22°C in minimal media containing galactose: SNY121-12A/pSB18 + pSL2099 (GRD20 END4), SBY4-10A/pSB1-125 + pSL2099 (grd20-1 END4), and SNY44-13B/pSL2099 (GRD20 end4-1). The cultures were then shifted to nonpermissive temperature (36°C), and 15 min later glucose was added to shut off expression of Ste3-myc. Equivalent volumes of culture were withdrawn at 0, 30, 60, and 90 min after addition of glucose. Protein extracts were immediately prepared from the cells, separated by SDS-PAGE, and analyzed by Western blotting using a mouse antic-myc antibody. The blots were developed as described in MATERIALS AND METHODS.
Figure 11
Figure 11
Grd20-HA partially colocalizes with the TGN marker A-ALP. Strain SBY4-10A carrying plasmids pSB10 and pAH16 was fixed, spheroplasted, and costained with anti-HA and anti-ALP antibodies. The anti-HA antibody was detected with an alexa 488-conjugated secondary antibody, whereas the anti-ALP antibody was detected with a Texas Red-conjugated secondary antibody. The cells were viewed by epifluorescence using filters specific for each fluorochrome. Two fields stained for each antigen are shown, with the panels on the right representing images derived from a merge of the images for each antigen.
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