Limits...
Mutants in trs120 disrupt traffic from the early endosome to the late Golgi.

Cai H, Zhang Y, Pypaert M, Walker L, Ferro-Novick S - J. Cell Biol. (2005)

Bottom Line: Transport protein particle (TRAPP), a large complex that mediates membrane traffic, is found in two forms (TRAPPI and -II).Surprisingly, we report that mutations in trs120 do not block general secretion.Furthermore, we demonstrate that Trs120p largely colocalizes with the late Golgi marker Sec7p.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06519, USA.

ABSTRACT
Transport protein particle (TRAPP), a large complex that mediates membrane traffic, is found in two forms (TRAPPI and -II). Both complexes share seven subunits, whereas three subunits (Trs130p, -120p, and -65p) are specific to TRAPPII. Previous studies have shown that mutations in the TRAPPII-specific gene trs130 block traffic through or from the Golgi. Surprisingly, we report that mutations in trs120 do not block general secretion. Instead, trs120 mutants accumulate aberrant membrane structures that resemble Berkeley bodies and disrupt the traffic of proteins that recycle through the early endosome. Mutants defective in recycling also display a defect in the localization of coat protein I (COPI) subunits, implying that Trs120p may participate in a COPI-dependent trafficking step on the early endosomal pathway. Furthermore, we demonstrate that Trs120p largely colocalizes with the late Golgi marker Sec7p. Our findings imply that Trs120p is required for vesicle traffic from the early endosome to the late Golgi.

Show MeSH

Related in: MedlinePlus

The analysis of invertase secretion in trs130 and -120 mutants. (A) A schematic diagram of trs120 and -130 mutants used in this study. The trs120-1 mutant was made by truncating 481 amino acids from the COOH terminus of Trs120p. The other trs120 mutants were made by transposon mutagenesis as described in Materials and methods. The arrows point to the position where the transposon is inserted in each mutant. Five trs130 mutants were made by truncating 33, 54, 74, 94, and 124 amino acids from the COOH terminus of Trs130p. Invertase secretion is blocked in all trs130 mutants (B) but not all trs120 mutants (C). Wild-type (WT) and mutant cells were grown at 25°C and preincubated at 37°C for 20 min. Cells were resuspended in low-glucose medium to induce the synthesis of invertase and were radiolabeled for 60 min. Samples were then processed for the immunoprecipitation of internal (I) and periplasmic (E) invertase. To facilitate the detection of the high molecular mass form of invertase in trs130 mutants, cells were transformed with the SUC2 gene on a 2-μm plasmid.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171297&req=5

fig1: The analysis of invertase secretion in trs130 and -120 mutants. (A) A schematic diagram of trs120 and -130 mutants used in this study. The trs120-1 mutant was made by truncating 481 amino acids from the COOH terminus of Trs120p. The other trs120 mutants were made by transposon mutagenesis as described in Materials and methods. The arrows point to the position where the transposon is inserted in each mutant. Five trs130 mutants were made by truncating 33, 54, 74, 94, and 124 amino acids from the COOH terminus of Trs130p. Invertase secretion is blocked in all trs130 mutants (B) but not all trs120 mutants (C). Wild-type (WT) and mutant cells were grown at 25°C and preincubated at 37°C for 20 min. Cells were resuspended in low-glucose medium to induce the synthesis of invertase and were radiolabeled for 60 min. Samples were then processed for the immunoprecipitation of internal (I) and periplasmic (E) invertase. To facilitate the detection of the high molecular mass form of invertase in trs130 mutants, cells were transformed with the SUC2 gene on a 2-μm plasmid.

Mentions: To further characterize the function of TRAPPII in membrane traffic, we constructed a collection of temperature-sensitive mutations in genes that encode essential TRAPPII-specific subunits. Initially, PCR mutagenesis was used to isolate mutants in trs120. However, after this approach failed to yield mutants, transposon mutagenesis was performed. The location of the transposon insertion in each of the seven mutants we isolated is shown in Fig. 1 A. An eighth mutant, trs120-1, was constructed by truncating the last 481 amino acids of Trs120p (Fig. 1 A). Further deletion of the COOH terminus of Trs120p led to cell death. Five truncation mutants in trs130 were also constructed. These mutants deleted the region of TRS130 that encodes the last 33–124 amino acids (Fig. 1 A). Deletion of TRS65, which encodes the third TRAPPII-specific subunit, did not affect the growth of yeast or membrane traffic (Sacher et al., 2001).


Mutants in trs120 disrupt traffic from the early endosome to the late Golgi.

Cai H, Zhang Y, Pypaert M, Walker L, Ferro-Novick S - J. Cell Biol. (2005)

The analysis of invertase secretion in trs130 and -120 mutants. (A) A schematic diagram of trs120 and -130 mutants used in this study. The trs120-1 mutant was made by truncating 481 amino acids from the COOH terminus of Trs120p. The other trs120 mutants were made by transposon mutagenesis as described in Materials and methods. The arrows point to the position where the transposon is inserted in each mutant. Five trs130 mutants were made by truncating 33, 54, 74, 94, and 124 amino acids from the COOH terminus of Trs130p. Invertase secretion is blocked in all trs130 mutants (B) but not all trs120 mutants (C). Wild-type (WT) and mutant cells were grown at 25°C and preincubated at 37°C for 20 min. Cells were resuspended in low-glucose medium to induce the synthesis of invertase and were radiolabeled for 60 min. Samples were then processed for the immunoprecipitation of internal (I) and periplasmic (E) invertase. To facilitate the detection of the high molecular mass form of invertase in trs130 mutants, cells were transformed with the SUC2 gene on a 2-μm plasmid.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171297&req=5

fig1: The analysis of invertase secretion in trs130 and -120 mutants. (A) A schematic diagram of trs120 and -130 mutants used in this study. The trs120-1 mutant was made by truncating 481 amino acids from the COOH terminus of Trs120p. The other trs120 mutants were made by transposon mutagenesis as described in Materials and methods. The arrows point to the position where the transposon is inserted in each mutant. Five trs130 mutants were made by truncating 33, 54, 74, 94, and 124 amino acids from the COOH terminus of Trs130p. Invertase secretion is blocked in all trs130 mutants (B) but not all trs120 mutants (C). Wild-type (WT) and mutant cells were grown at 25°C and preincubated at 37°C for 20 min. Cells were resuspended in low-glucose medium to induce the synthesis of invertase and were radiolabeled for 60 min. Samples were then processed for the immunoprecipitation of internal (I) and periplasmic (E) invertase. To facilitate the detection of the high molecular mass form of invertase in trs130 mutants, cells were transformed with the SUC2 gene on a 2-μm plasmid.
Mentions: To further characterize the function of TRAPPII in membrane traffic, we constructed a collection of temperature-sensitive mutations in genes that encode essential TRAPPII-specific subunits. Initially, PCR mutagenesis was used to isolate mutants in trs120. However, after this approach failed to yield mutants, transposon mutagenesis was performed. The location of the transposon insertion in each of the seven mutants we isolated is shown in Fig. 1 A. An eighth mutant, trs120-1, was constructed by truncating the last 481 amino acids of Trs120p (Fig. 1 A). Further deletion of the COOH terminus of Trs120p led to cell death. Five truncation mutants in trs130 were also constructed. These mutants deleted the region of TRS130 that encodes the last 33–124 amino acids (Fig. 1 A). Deletion of TRS65, which encodes the third TRAPPII-specific subunit, did not affect the growth of yeast or membrane traffic (Sacher et al., 2001).

Bottom Line: Transport protein particle (TRAPP), a large complex that mediates membrane traffic, is found in two forms (TRAPPI and -II).Surprisingly, we report that mutations in trs120 do not block general secretion.Furthermore, we demonstrate that Trs120p largely colocalizes with the late Golgi marker Sec7p.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06519, USA.

ABSTRACT
Transport protein particle (TRAPP), a large complex that mediates membrane traffic, is found in two forms (TRAPPI and -II). Both complexes share seven subunits, whereas three subunits (Trs130p, -120p, and -65p) are specific to TRAPPII. Previous studies have shown that mutations in the TRAPPII-specific gene trs130 block traffic through or from the Golgi. Surprisingly, we report that mutations in trs120 do not block general secretion. Instead, trs120 mutants accumulate aberrant membrane structures that resemble Berkeley bodies and disrupt the traffic of proteins that recycle through the early endosome. Mutants defective in recycling also display a defect in the localization of coat protein I (COPI) subunits, implying that Trs120p may participate in a COPI-dependent trafficking step on the early endosomal pathway. Furthermore, we demonstrate that Trs120p largely colocalizes with the late Golgi marker Sec7p. Our findings imply that Trs120p is required for vesicle traffic from the early endosome to the late Golgi.

Show MeSH
Related in: MedlinePlus