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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.

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Trs120p resides on a late Golgi/early endosomal compartment. Lysates were prepared and fractionated on a sucrose density gradient as described in Materials and methods. Trs120p does not colocalize with the early Golgi marker Och1p (A) or the ER-Golgi SNARE Sec22p (B). Trs120p largely cofractionates with the late Golgi/early endosomal marker Chs3p (C).
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fig7: Trs120p resides on a late Golgi/early endosomal compartment. Lysates were prepared and fractionated on a sucrose density gradient as described in Materials and methods. Trs120p does not colocalize with the early Golgi marker Och1p (A) or the ER-Golgi SNARE Sec22p (B). Trs120p largely cofractionates with the late Golgi/early endosomal marker Chs3p (C).

Mentions: In an attempt to better resolve the different compartments of the Golgi by subcellular fractionation, we modified the sucrose density gradient we had previously used. In earlier studies, a step gradient containing 11 different concentrations of sucrose, ranging from 18 to 60%, was used. The TRAPPII complex fractionated between 26 and 48% sucrose on this gradient (Sacher et al., 2001). For this paper, we designed a new step gradient containing 11 1-ml steps that ranged from 26 to 50% sucrose (see details in Materials and methods). Och1p, which marks the earliest known carbohydrate-modifying compartment in the yeast Golgi, was used as an early Golgi marker (Nakayama et al., 1992; Brigance et al., 2000), whereas Chs3p marked both the late Golgi and early endosomes (Valdivia et al., 2002). Late Golgi and early endosomes are difficult to resolve by sucrose density analysis (Holthuis et al., 1998; Valdivia et al., 2002). Thus, it is unclear whether late Golgi markers solely mark this compartment or if they also mark the early endosome. With our gradient conditions, Och1p peaked in fraction 4 (Fig. 7 A), whereas Chs3p peaked in fraction 8 (Fig. 7 C). The SNARE Sec22p peaked in fractions 3 and 11 (Fig. 7 B). The later peak (at 47% sucrose) represents the fraction of Sec22p that resides on the ER, whereas the earlier peak (at 29% sucrose) is Golgi-bound Sec22p (Barrowman et al., 2000). This result confirms that our new gradient conditions clearly resolve early and late Golgi subcompartments. To monitor the localization of TRAPPII on this gradient, we added a 13-x-myc tag to the genomic copy of Trs120p as described previously (Longtine et al., 1998). Tagged Trs120p is functional, as the presence of the tag did not impede growth or the assembly of the TRAPPII complex. Trs120p largely cofractionated with Chs3p (Fig. 7 C) but not with Och1p and Sec22p (Fig. 7, A and B). These findings imply that Trs120p resides on a late Golgi/early endosomal compartment that is marked by Chs3p.


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)

Trs120p resides on a late Golgi/early endosomal compartment. Lysates were prepared and fractionated on a sucrose density gradient as described in Materials and methods. Trs120p does not colocalize with the early Golgi marker Och1p (A) or the ER-Golgi SNARE Sec22p (B). Trs120p largely cofractionates with the late Golgi/early endosomal marker Chs3p (C).
© Copyright Policy
Related In: Results  -  Collection

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

fig7: Trs120p resides on a late Golgi/early endosomal compartment. Lysates were prepared and fractionated on a sucrose density gradient as described in Materials and methods. Trs120p does not colocalize with the early Golgi marker Och1p (A) or the ER-Golgi SNARE Sec22p (B). Trs120p largely cofractionates with the late Golgi/early endosomal marker Chs3p (C).
Mentions: In an attempt to better resolve the different compartments of the Golgi by subcellular fractionation, we modified the sucrose density gradient we had previously used. In earlier studies, a step gradient containing 11 different concentrations of sucrose, ranging from 18 to 60%, was used. The TRAPPII complex fractionated between 26 and 48% sucrose on this gradient (Sacher et al., 2001). For this paper, we designed a new step gradient containing 11 1-ml steps that ranged from 26 to 50% sucrose (see details in Materials and methods). Och1p, which marks the earliest known carbohydrate-modifying compartment in the yeast Golgi, was used as an early Golgi marker (Nakayama et al., 1992; Brigance et al., 2000), whereas Chs3p marked both the late Golgi and early endosomes (Valdivia et al., 2002). Late Golgi and early endosomes are difficult to resolve by sucrose density analysis (Holthuis et al., 1998; Valdivia et al., 2002). Thus, it is unclear whether late Golgi markers solely mark this compartment or if they also mark the early endosome. With our gradient conditions, Och1p peaked in fraction 4 (Fig. 7 A), whereas Chs3p peaked in fraction 8 (Fig. 7 C). The SNARE Sec22p peaked in fractions 3 and 11 (Fig. 7 B). The later peak (at 47% sucrose) represents the fraction of Sec22p that resides on the ER, whereas the earlier peak (at 29% sucrose) is Golgi-bound Sec22p (Barrowman et al., 2000). This result confirms that our new gradient conditions clearly resolve early and late Golgi subcompartments. To monitor the localization of TRAPPII on this gradient, we added a 13-x-myc tag to the genomic copy of Trs120p as described previously (Longtine et al., 1998). Tagged Trs120p is functional, as the presence of the tag did not impede growth or the assembly of the TRAPPII complex. Trs120p largely cofractionated with Chs3p (Fig. 7 C) but not with Och1p and Sec22p (Fig. 7, A and B). These findings imply that Trs120p resides on a late Golgi/early endosomal compartment that is marked by Chs3p.

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