Multiple cytosolic and transmembrane determinants are required for the trafficking of SCAMP1 via an ER–Golgi–TGN–PM pathway

Abstract

SUMMARYHow polytopic plasma membrane (PM) proteins reach their destination in plant cells remains elusive. Using transgenic tobacco BY-2 cells, we previously showed that the rice secretory carrier membrane protein 1 (SCAMP1), an integral membrane protein with four transmembrane domains (TMDs), is localized to the PM and trans-Golgi network (TGN). Here, we study the transport pathway and sorting signals of SCAMP1 by following its transient expression in tobacco BY-2 protoplasts and show that SCAMP1 reaches the PM via an endoplasmic reticulum (ER)-Golgi-TGN-PM pathway. Loss-of-function and gain-of-function analysis of various green fluorescent protein (GFP) fusions with SCAMP1 mutations further demonstrates that: (i) the cytosolic N-terminus of SCAMP1 contains an ER export signal; (ii) the transmembrane domain 2 (TMD2) and TMD3 of SCAMP1 are essential for Golgi export; (iii) SCAMP1 TMD1 is essential for TGN-to-PM targeting; (iv) the predicted topology of SCAMP1 and its various mutants remain identical as demonstrated by protease protection assay. Therefore, both the cytosolic N-terminus and TMD sequences of SCAMP1 play integral roles in mediating its transport to the PM via an ER-Golgi-TGN pathway.

The Golgi-Localized Arabidopsis Endomembrane Protein12 Contains Both Endoplasmic Reticulum Export and Golgi Retention Signals at Its C Terminus

“…By contrast, GFP-EMP12 was largely separated from the TGN marker mRFP-SYP61 and the PVC marker mRFP-VSR2 in Arabidopsis protoplasts ( Figures 4B and 4C). In addition, when the ER-to-Golgi traffic was blocked by overexpression of Sec12p, a Sar1p-specific guanosine nucleotide exchange factor for COPII vesicle formation (Pimpl et al, 2003;Cai et al, 2011), both GFP-EMP12 and Man1-mRFP were trapped and colocalized within the ER ( Figure 4D), indicating that GFP-EMP12 reached the Golgi via the COPII-dependent ER-to-Golgi sorting route, a result consistent with the Golgi localization of Man1-mRFP (Langhans et al, 2008).…”

“…The N terminus of GFP-EMP12 was found to be resistant to protease digestion because the 47-kD GFP-TMD1 fragment remained intact in the presence of trypsin, whereas the 75-kD full-length GFP-EMP12 fusion did not ( Figure 1C, lanes 1 and 2), indicating the lumenal location of the GFP tag as predicted ( Figure 1A). The reliability of such a protease protection assay was further verified by an identical experiment using a GFP fusion of the type I integral membrane protein GFP-VSR2 as a control, in which the lumenal GFP remained intact upon protease digestion ( Figure 1C, lanes 4 and 5; Cai et al, 2011).…”

“…GFP fusions with the SCAMP1 (SCAMP1-GFP or GFP-SCAMP1), an integral membrane protein with four TMDs, were localized to both the TGN and PM via an ER-Golgi-TGN-PM pathway (Lam et al, 2007b;Cai et al, 2011). Indeed, the original TGN/PM localization of SCAMP1-GFP was altered by the addition of the RNIKCD motif to the C terminus of SCAMP1-GFP because SCAMP1-GFP-RNIKCD was largely colocalized with the Golgi marker Man1-mRFP ( Figure 12A, panels 1 and 2) in Arabidopsis protoplasts.…”

“…Microsome isolation and protein topology analysis were performed as described with some modifications (Abas and Luschnig, 2010;Cai et al, 2011). Basically, the protoplasts expressing GFP-EMP12 or GFP-VSR2 were diluted with 250 mM NaCl and harvested by centrifugation.…”

“…More recently, the rice (Oryza sativa) SECRETORY CARRIER MEMBRANE PRO-TEIN1 (SCAMP1), a polytopic PM protein with four TMDs (Lam et al, 2007a(Lam et al, , 2007b(Lam et al, , 2008Law et al, 2012), was shown to reach the PM via an ER-Golgi-TGN-PM pathway in tobacco (Nicotiana tabacum) Bright Yellow-2 cells (Cai et al, 2011). Interestingly, both its cytosolic sequences and TMDs regulated the proper trafficking steps from ER to PM owing to the presence of various signals within the SCAMP1, including the ER export signal in the cytosolic N terminus, the Golgi export signal within the TMD2-TMD3, and the TGN-PM targeting signal in TMD1 (Cai et al, 2011). However, the underlying mechanisms remain elusive.…”