BP-80 and Homologs are Concentrated on Post-Golgi, Probable Lytic Prevacuolar Compartments

Abstract

Prevacuolar compartments (PVCs) are membrane-bound organelles that mediate protein traffic between Golgi and vacuoles in the plant secretory pathway. Here we identify and define organelles as the lytic prevacuolar compartments in pea and tobacco cells using confocal immunofluorescence. We use five different antibodies specific for a vacuolar sorting receptor (VSR) BP-80 and its homologs to detect the location of VSR proteins. In addition, we use well-established Golgi-markers to identify Golgi organelles. We further compare VSR-labeled organelles to Golgi organelles so that the relative proportion of VSR proteins in Golgi vs. PVCs can be quantitated. More than 90% of the BP-80-marked organelles are separate from Golgi organelles; thus, BP-80 and its homologs are predominantly concentrated on the lytic PVCs. Additionally, organelles marked by anti-AtPep12p (AtSYP21p) and anti-AtELP antibodies are also largely separate from Golgi apparatus, whereas VSR and AtPep12p (AtSYP21p) were largely colocalized. We have thus demonstrated in plant cells that VSR proteins are predominantly present in the lytic PVCs and have provided additional markers for defining plant PVCs using confocal immunofluorescence. Additionally, our approach will provide a rapid comparison between markers to quantitate protein distribution among various organelles.

Rice SCAMP1 Defines Clathrin-Coated,trans-Golgi–Located Tubular-Vesicular Structures as an Early Endosome in Tobacco BY-2 Cells

“…A rhodamine-conjugated Fab fragment then was added and incubated at room temperature for 4 h before a second wash, followed by the addition of the second primary antibody. Additionally, we confirmed that the labeling pattern for an antibody used individually matched the pattern obtained with the same antibody when used in double labeling experiments (Jiang et al, 2000;Li et al, 2002). Secondary antibodies alone were also used as an additional control.…”

“…Fixation and preparation of tobacco BY2 cells and their labeling and analysis by epifluorescence and confocal immunofluorescence have been described previously (Jiang and Rogers, 1998;Jiang et al, 2000;Li et al, 2002;Tse et al, 2004). The settings for collecting confocal images within the linear range were as described (Jiang and Rogers, 1998).…”

“…The AM4-64-treated cells were then washed with ice-cold MS medium several times and transferred to room temperature for uptake. During the incubation period, cells were collected at the indicated time intervals for chemical fixation and confocal immunofluorescence with antibodies against VSR and SCAMP according to procedures described previously (Jiang and Rogers, 1998;Jiang et al, 2000;Li et al, 2002;Tse et al, 2004).…”

“…Recent investigations in tobacco (Nicotiana tabacum) BY-2 cells and Arabidopsis protoplasts have shown that PVCs are enriched in VSR proteins and are also characterized by the presence of Rha1, a Rab5 homolog, the t-SNARE Pep12p, and plant retromer homologs (Li et al, 2002;Sohn et al, 2003;Tse et al, 2004;Oliviusson et al, 2006). These organelles have a typical multivesiculate morphology (Tse et al, 2004;Mo et al, 2006;Oliviusson et al, 2006).…”

Dynamic Response of Prevacuolar Compartments to Brefeldin A in Plant Cells

“…Putative plant PVCs were identified using antibodies specific for the Arabidopsis (Arabidopsis thaliana) syntaxin Pep12p and the pea (Pisum sativum) vacuolar sorting receptor (VSR) BP-80 (Bassham et al, 1995;Conceicao et al, 1997;Paris et al, 1997). Studies on the relative distribution of VSR proteins between Golgi stacks and PVCs in various plant cells demonstrated that VSR proteins largely separated from Golgi markers and thus must be concentrated on PVCs under steady-state conditions (Li et al, 2002). In addition, a chimeric BP-80 reporter containing the BP-80 transmembrane domain and cytoplasmic tail colocalized with endogenous VSR proteins when the reporter was expressed in transgenic tobacco (Nicotiana tabacum) culture cells (Jiang and Rogers, 1998).…”